Marine Biological Laboratory Library Woods Hole, Mas3achusetl5 Physiology of the Fungi McGRAW-HILL PUBLICATIONS IN THE BOTANICAL SCIENCES Edmund W. Sinnott, Consulting Editor ARNOLD An Introduction to Paleobotany CURTIS AND CLARK An Introduction to Plant Physiology EAMES Morphology of the Angiosperms EAMES Morphology of Vascular Plants: Lower Groups EAMES AND MACDANiELS An Introduction to Plant Anatomy HAUPT An Introduction to Botany HAUPT Laboratory Manual of Elementary Botany HAUPT Plant Morphology HILL Economic Botany HILL, OVERHOLTS, POPP, AND GROVE Botauy JOHANSEN Plant Microtechnique KRAMER Plant and Soil Water Relationships KRAMER AND KOZLOWSKi Physiology of Trecs LILLY AND BARNETT PhvsiologV of the Fungi MAHESHWARi An Introduction to the Embryology of the Angiosperms MILLER Plant Physiology POOL Flowers and Flowering Plants SHARP Fundamentals of Cytology SINNOTT Plant Morphogenesis SINNOTT, DUNN, AND DOBZHANSKY Principles of Genetics SINNOTT AND WILSON Botany: Principles and Problems SMITH Cryptogamic Botany Vol. I. Algae and Fungi Vol. II. Bryophytes and Pteridophytes SMITH The Fresh-water Algae of the United States SWINGLE Textbook of Systematic Botany WEAVER AND CLEMENTS Plant Ecology There are also the related series of McGraw-Hill Publications in the Zoological Sciences, of which E. J. Boell is Consulting Editor, and in the Agricultural Sciences, of which R. A. Brink is Consulting Editor. Frontispiece. Pilobolus showing phototropism. See page 339. L -VI Physiology of the Fungi VIRGIL GREENE LILLY Professor of Physiology, Department of Plant Pathology and Bacteriology, West Virginia University; Physiologist, West Virginia Agricultural Experiment Station HORACE L. BARNETT Professor of Mycology, Department of Plant Pathology and Bacteriology, West Virginia University; Mycologist, West Virginia Agricultural Experiment Station 1951 McGRAW-HILL BOOK COMPANY New York Toronto London PHYSIOLOGY OF THE FUNGr Copyright, 1951, by the McGraw-Hill Book Company, Inc. Prmtedinthe United States of America. All rights reserved. This book, or parts thereof, may not be reproduced in any form without permission of the pubUshers. 7 89 10 11 12-MAMM-l 09 8 37865 This book is dedicated to the mem- ory of Leon H. Leonian and to Ernst A. Bessey. The guidance and inspiration of these men in directing our interests to the study of fungi is gratefully acknowledged. PREFACE Living fungi are being studied more intensively than ever before. This may be attributed in part to increased interest in the potentiahties of the fungi in industry as well as to the greater recognition of fungi as important disease-producing agents of plants and animals and as destroy- ers of fabrics and other cellulosic materials of commercial importance. This has increased the interest in the cultivation of the fungi and has shown the need for an adequate textbook covering the broad aspects of physiology of the fungi, their growth requirements, and activities. It was the intent of the authors to prepare a textbook which would fulfill the needs of students desirous of some training in this field. This book is primarily a text for the advanced student and assumes some basic knowledge of the morphology of fungi and of organic chemistry. It had its origin in the lectures and laboratory exercises used for three years by the authors in a course in physiology of the fungi offered to graduate students at West Virginia University. The authors have contributed equally of their time and efforts in the preparation of this text. For those who are interested or are actively engaged in physiological research on fungi, this textbook may serve as a reference book and as an entry into the literature. The large ever-growing accumulation of liter- ature has also made it desirable to bring together a summary and dis- cussion of the information in this field. However, no attempt has been made at complete documentation of the subjects discussed. Certain particularly important references are marked with a star and are recom- mended as required reading for students. For the most part, the scientific names of the fungi are those which were used by the investigators whose work has been cited. No attempt has been made to reduce these names to synonomy. Because of the close relation between fungus physiology and plant pathology, plant pathogenic fungi have been used as examples whenever possible. Several suggested laboratory exercises with suggested test fungi are included at the end of the text, so that other teachers might profit by the authors' experience in designing and conducting laboratory work in fungus physiology. All tables, graphs, and photographs not credited to other sources are original. It is a pleasure to acknowledge our indebtedness to the many individuals ix X PREFACE who have aided us with their suggestions. Among our colleagues at West Virginia University who have read portions of the manuscript are: J. G. Leach, C. R. Orton, M. E. Gallegly, R. P. True, W. T. Jackson, B. G. Anderson, R. B. Dustman, and J. H. Hare. In addition, the following have read one or more chapters: B. W. Henry, J. B. Routien, W. D. Gray, and J. B. Conn. We are also indebted to Mrs. H. L. Barnett, who typed most of the manuscript. We wish to thank the many individuals, societies, and publishers who have granted us permission to use data and illustrations. Particular acknowledgment is made in connection with the material cited. Virgil Greene Lilly Horace L. Barnett MORGANTOWN, W. Va, May, 1951 CONTENTS Preface ix 1. Introduction 1 Fungus Physiology in Relation to Other Sciences — Aims — Scope — Historical Development. 2. Culture Media 8 ICinds of Media — Natural Versus Synthetic Media — Choice and Preparation of Media — Ways of Expressing Concentration — Comparison of Media — - Summary. 3. Growth 24 Phases of Growth — Rate of Growth — Ways of Measuring Growth — Methods of Presenting Results — Factors Affecting Growth — Effect of External Factors on Morphology — Summary. 4. Enzymes and Enzyme Action 45 Classification of Enzymes — Chemical Nature of Enzymes — Factors Affecting Enzyme Activity — Mechanism of Enzyme Action — Adaptive Enzymes- Energy and Energy Utilization by Fungi — Summary. 5. Essential Metallic Elements 65 Biological Essential Elements — The Essential Macro Elements — Essential Micro Elements — Periodicity of Biologically Essential Elements — Summary. 6. The Essential Xonmetallic Elements Other Than Carbon 87 Hydrogen — Oxygen — Sulfur — Phosphorus — Nitrogen — Other Nonmetallic Elements — Summary. 7. Carbon Sources and Carbon Utilization 116 Monosaccharides and Related Compounds — Organic Acids — Glycosides — Oligosaccharides — Polysaccharides — Heterotrophic Utilization of Carbon Dioxide — Utilization of Carbon — Summary. 8. Hydrogen-ion Concentration 149 Ionization of Compounds — The Meaning of pH — Buffers and Buffer Capacity — Methods of Determining pH Values — Effects on Fungi — Summary. 9. Vitamins and Growth Factors 171 Part I. General Considerations — Synthesis of Vitamins by Fungi — Vitamin xi xii CONTENTS Deficiencies in Fungi — Inhibitory Effects of Vitamins — Vitamers — Unidenti- fied Growth Factors, Part II. Specific Vitamins — Thiamine and Its Moieties — Biotin — Inositol — Nicotinic Acid — Pantothenic Acid — Pyridoxine — p-Aminobenzoic Acid — Riboflavin — Summary. 10. Fungi AS Test Organisms 208 General Procedures — Vitamin Assays — Amino-acid Assays — Assays for Essential Elements — Sugars — Tests for Certain Metabolic Products — Testing Fabric Protectants — Summary. 11. Metabolic Antagonists 226 Antivitamins — Amino-acid Antagonists — Development of Fastness — Sum- mary. 12. The Action of Fungicides 245 Copper — Mercury — Sulfur — Organic Fungicides — Evaluating Fungicides — Summary. 13. Metabolic Products 266 Decomposition of Organic Materials — Fungi as Food — Cultivation of Fungi for Food — Fat Production — Production of Vitamins — Enzyme Production — Alcoholic Fermentation — Organic Acids — Esters — Antibiotics and Drugs — Toxins — Pigments — Summary. 14. Factors Influencing Sporulation op Fungi 304 Environmental Factors — Other Physical Factors — Nutritional Factors — Other Factors — Summary. 15. Spore Discharge and Dissemination 338 Methods of Spore Discharge — Influence of External Conditions — Spore Dissemination — Summary. 16. Spore Germination 355 Physical Factors — Nutrients and Stimulants — Longevity of Spores — Sum- mary. 17. The Physiology of Parasitism and Resistance 372 Penetration — Parasitism — Resistance — Summ ary . 18. Physiological Variation and Inheritance of Physiological Char- acters 400 Physiological Variation — Inheritance of Physiological Characters — Summary. Suggested Laboratory Exercises 419 Index 441 CHAPTER 1 INTRODUCTION The primary role of the fungi in nature has been fittingly described in the prophetic statement of B. O. Dodge (1939) : . . . the fungi are not degenerate organisms which are on their way out in a scheme of evolution, and so of little economic importance and scientific interest. The fungi, on the contrary, are progressive, ever changing and evolving rapidly in their own way so that they are capable of becoming readily adapted to every condition of life. We may rest assured that as green plants and animals disappear one by one from the face of the globe, some of the fungi will always be present to dispose of the last remains. The most important role of the fungi in the economy of nature is to act as scavengers in disposing of dead and fallen vegetation. In this way the biologically essential elements are released for reuse, and the balance of nature is maintained. However, these are not the only functions of the fungi which are of interest and importance to man. Since the beginning of agriculture fungi have been used to prepare bread and other foods, as well as fermented beverages. Some fungi cause diseases of plants and animals. Knowledge of their role as the causal agents of plant dis- eases long antedated the recognition of bacterial diseases. While yeasts have long been used to produce alcohol, the vast potentialities of other species for the industrial production of organic acids and antibiotics have been recognized more recently. An understanding of life processes of the fungi is essential whether one wishes to control the fungi which cause disease, to employ them in industry, or to use them in the laboratory to unlock the secrets of nature. The domain of physiology is the study of functions or life processes. Fungus physiology is the study of living fungi, their functions and ac- tivities, how they affect their environment and how the environment affects them. Like other branches of science, fungus physiology has four phases of development: (1) the discovery and verification of facts, which are the foundation of any science, (2) the organization of these facts into a systematic and coherent body of knowledge, (3) the dissemina- tion of newly discovered facts, and (4) use of the newly discovered facts and others already known to formulate principles. Facts are the basis of science, but facts alone are sterile unless they are seen in relation to 1 2 PHYSIOLOGY OF THE FUNGI previous knowledge. Organization and interpretation of facts are equally as important as the experimentation which reveals them. The fungi as a group are highly responsive to their environment and are thus excellent test organisms for inquiring into the secrets of nature. Nature always answers correctly the questions we ask, and, in this sense, no experiment is a failure, although we may fail to ask the question we intended, or we may misunderstand the answer given. Infinite care is required to frame a question so that a definite answer may be obtained. By observing fungi in nature we are limited to questions asked by nature. Commonly, the environmental and nutritional factors are so complex that the influence of a single variable cannot be evaluated. By controlling the conditions under which a fungus is placed in the laboratory it is possible to ask questions of great precision. Indeed, the number and scope of the questions which we may ask fungi are limited only by the present-day techniques and the curiosity of the investigator. Since most of our knowledge of the physiology of the fungi has been gained from laboratory investigations, the experimental approach will be emphasized in the discussions which follow. However, this choice is not meant to minimize the importance of and need for critical observa- tions in nature. By emphasizing the results of careful laboratory re- search, we are better able in the following chapters to present the facts necessary for an understanding of the vital principles of fungus physi- ology, and also to show that these principles, theories, and hypotheses are founded upon experimental evidence. FUNGUS PHYSIOLOGY IN RELATION TO OTHER SCIENCES Physiology is that branch of science which deals with the life processes or the activities of organisms. The activities of the whole organism or of any of its parts may be hmited by its form or structure. Both the activity and the form of an individual are determined to a great extent by its genetic constitution and are modified by the environment to which the organism is exposed. Physiology, therefore, is not an independent subject. An understanding of physiological principles is based, in part, upon facts and theories from many other fields of science, such as chem- istry, physics, anatomy, cytology, bacteriology, and genetics. Many of the physiological principles which have been established for one group of organisms apply equally well to other groups. The vita- mins essential to the normal growth of the fungi are the same as those required by man, animals, and the higher plants. The general functions of these vitamins appear to be the same in all organisms. The differ- ence in the vitamin requirements seems to lie in the different abilities of these groups of organisms (or individuals within the group) to synthesize these necessary compounds. As Schopfer (1943) has pointed out, the INTRODUCTION 3 vitamin problem is common to many branches of science. Many other problems investigated in fungus physiology are likewise common to other related fields of study. In a similar way, a better understanding of certain related fields is gained by knowledge of fungus physiology. The plant pathologist com- monly finds it necessary to study the living parasitic fungus apart from its host and must know something of the cultural methods and the spe- cific nutritional requirements of the fungus at hand. The mycologist and plant pathologist are faced with numerous unsolved problems which must be investigated by physiological methods. One of the most chal- lenging problems is the cultivation of certain fungi now classed as obligate parasites on synthetic media of known composition. Until this is accom- plished, the nutritional requirements of these fungi cannot be fully deter- mined. Such knowledge would without doubt lead to a better under- standing of parasitism and resistance. The taxonomic mycologist uses morphological characters almost exclu- sively in his identification and classification studies, while the bacteriol- ogist, being unable to use distinct morphological features to any great extent, emphasizes the physiological characters in classifying bacteria. Much more information is needed before it can be determined whether any physiological characters are sufficiently valuable and uniform to be used to supplement morphological characters in taxonomy of fungi. It seems logical that such physiological differences between groups of fungi do exist, and that the main problem lies in the discovery and recognition of these characters and their application to taxonomy. On the other hand, caution must be observed, for nutritional and environmental con- ditions are known to affect, to a certain extent, some morphological characters used in classification. The geneticist and the biochemist may find the fungi interesting and suitable subjects for the study of their respective problems, while the bacteriologist finds many points of similarity between the physiology of the bacteria and that of the fungi. Industry has used many species of fungi to its ovnx advantage for many decades. Yeasts were used long before the physiology of the fungi became an organized study, but the search for superior strains of yeast continues. The widespread use of antibiotics has brought under laboratory study many species of fungi which would otherwise have been ignored. This has created many new problems of nutrition, especially with regard to large-scale cultivation of these fungi. Thus, knowledge of the life activities of the fungi is important and useful in many related fields of science, just as some knowledge of these related fields is essential to an understanding of the fimgi. The study of fungup physiology is justified as a separate field in which the basic or fundamental 4 PHYSIOLOGY OF THE FUNGI principles are the aim, or as a study closely integrated with the fields of science concerned with more practical problems. Often, the most \aluablc results are obtained when research is not restricted by the boundaries of practical application. AIMS This book is a discussion of living fungi, of their life processes and the factors which influence them. It is written primarily for the student who is acquainted with the structure of fungi and who is beginning the study of their activities. From the discussions which follow the student should gain a knowledge and understanding of the basic principles of fungus physiology. To this end a considerable amount of factual material con- cerning the behavior of specific fungi under specific conditions is cited. The secondary aim of this book is to present a limited number of selected references which may be of use to the student or investigator who wishes more detailed information. Where possible, review articles have been included. Complete documentation is impossible because of the tre- mendous volume of literature. However, becoming familiar with the literature is an essential part of a student's education. SCOPE As a text this book must cover many phases of the subject. One of the first problems to be considered is the choice and preparation of suitable media for growth and sporulation of the fungi under study. Since there is no universal medium suitable for all fungi, a wise choice of media for the purpose at hand is of fundamental importance in any investigation. Before a fungus can be studied in any great detail in the laboratory, it is necessary to determine the conditions which affect growth. Growth is a complex phenomenon, and some discussion of the phenomenon itself and the ways of measuring growth is necessary for the understanding of these conditions. Nutritional factors, such as source of nitrogen, source of carbon, the presence of essential elements and vitamins, and the pH of the substrate, afTect growth in interrelated ways. Each of these factors and its importance in growth and other activities of fungi are discussed at some length. The life processes of the fungi involve numerous chemical transforma- tions. Living organisms make and use special organic catalysts, enzymes, which control these reactions. The actions of the enzymes in the living organism are coordinated and interrelated. A knowledge of the princi- ples of enzyme action is essential to the study of fungus physiology. The fungi are able to make a far greater contribution to the production of food and many other valuable products than they do at present. Both INTRODUCTION 5 the useful metabolic products, such as alcohols, organic acids, and anti- biotics, and the harmful products (toxins) are discussed at some length. Certain fungi cause diseases of plants and animals. The action of fimgicides used to control these pathogens will be discussed from a theo- retical viewpoint, since there is an enormous amount of literature dealing with the practical application of fungicides. Too little attention has been devoted to the mechanism of fungicidal action. The production of spores, which is of fundamental importance to the fungus in the perpetuation of the species, affords many interesting prob- lems in fungus physiology. Environmental and nutritional factors play important roles in determining whether a fungus will spoiiilate under a given set of conditions. These factors are discussed in some detail. The latter portion of the text emphasizes the activities of fungi in nature. These topics include the discharge, dissemination, and germina- tion of spores and the physiological aspects of parasitism, variation, and inheritance. The physiology of parasitism and resistance is of special interest to plant pathologists and medical mycologists. Most of the examples are taken from the field of plant pathology. Perhaps the dis- cussion of these problems will stimulate the interest and curiosity of the student. A better understanding of parasitism will surely lead to a wiser choice of control methods for certain fungi. No study of fungus physiology is complete without experimental work in the laboratory. The judgment necessary to evaluate one's own work is founded upon experience. Suggested laboratory exercises and demon- strations, with brief instructions, are given at the end of the text. These are selected to illustrate important principles, many of which can be illustrated clearly only by direct observation of the varied reactions of fungi to their environment. HISTORICAL DEVELOPMENT The development of fungus physiology is far from complete. While some of the main outlines are clearly visible, much remains to be done. Although space and time do not permit a complete review of the history of this science, it is important to realize that its development was the work of many minds and hands. The influence of the early investigators continues, not only in their published work but also in the students they trained. Some of the outstanding leaders in the development of fungus physi- ology are Avorthy of special mention. Their names and their contribu- tions are encountered frequently bj'" all students of this subject. Brief mention of some of these men and their fields of interest and investigation is made below. 6 PHYSIOLOGY OF THE FUNGI Louis Pasteur (1822-1895), France. Pasteur was a chemist who, as a result of his interest in microorganisms which cause disease and fermenta- tions, became a biologist. No other scientist has opened up so many fields of fruitful study. Early in his career he discovered that fungi are able to discriminate between the optical isomers of tartaric acid. His student Raulin devised the first synthetic medium for the cultivation of fungi and published the first thorough study of the nutritional require- ments of a fungus. Pasteur discovered that some organisms are inhibited by free oxygen and that some fungi change both their morphology and physiology when cultivated anaerobically. Pasteur's complete works have been collected and edited (1933-1939) by his grandson, Professor Pasteur Vallery-Radot. Dubos (1950) has published an evaluation of Pasteur's work. Heinrich Anton de Bary (1831-1888), Germany. His principal contribu- tions to mycology dealt with life histories and parasitism of fungi. His interests were primarily with biological adaptations and were more physi- ological than taxonomic. De Bary's influence as a teacher attracted many students who later were responsible for much of the development of plant pathology and mycology. Among his writings was " Morphologic und Physiologic der Pilze" (first edition 1866, second edition, English translation, 1887), which may be considered as the first book containing discussions of the physiology of the fungi. Oscar Brefeld (1839-1925), Germany. We owe a great debt to this patient investigator, who developed methods of ensuring sterile media and apparatus for pure culture work. He was equally insistent with regard to the purity of his cultures. His chief interest in mycology was the study of life histories and development of fungi. This meant to him observation of a fungus from ''Spore zu Spore." He was the first to use the single-spore technique. Besides his occasional papers, he published his monumental work (1872-1912) in 15 parts. This beautifully illus- trated work is still of great value. Georg Klehs (1857-1918), Germany. His important contributions to the study of fungus physiology concerned problems related to sporulation. In 1900 he summarized his conclusions in four statements or laws (Chap. 14). No better generalizations on this subject have appeared in the 50 years which have elapsed since they were published. For an evaluation of the significance of Klebs' work, see Kauffman (1929). A. H. Reginald Buller (1874-1944), England and Canada. Many of his studies involved the activities of fungi in relation to structure. His chief interests lay in production of fruit bodies and spores, in spore discharge and dissemination, and in the effects of the environment on these activi- ties. His keen observations are recorded in detail in seven volumes, "Researches on Fungi." These volumes are written in an interesting, INTRODiCTION 7 readable style and should be frequently consulted by all students of mycology. Leon H. Leonian (1888-1945), United States. Trained as a mycologist under Kauffman, he was always interested in discovering the potentiali- ties of living fungi. His principal contributions were made in the studj'- of fungus nutrition with emphasis on the factors which are required by fungi for growth and reproduction. For a bibliography of his papers see Orton (1946). The number of living investigators who have made and are continuing to make important contributions to fungus physiology is far too great to list here, and for this reason they have been omitted. An idea of the scope of their interests and activities may be gained from the references in the following chapters. REFERENCES Bkefeld, O.: Botanische Untersuchungen iiber Schimmelpilze, Hefte 1-4, 1872- 1881. Title changed to Botanische Untersuchungen iiber Hefenpilze Fortset- zung der Schimmelpilze for Heft 5, 1883; thereafter Untersuchungen aus dem Gesamtgebiet der Mykologie, Hefte 6-15, 1884-1912. Hefte 1-8, Arthur Felix, Leipzig. Hefte 9-15, Heinrich Schoningh, Muenster. BuLLER, A. H. R.: Researches on Fungi, Longmans, Roberts and Green, London. Vol. I, 1909; Vol. II, 1922; Vol. Ill, 1924; Vol. IV, 1931; Vol. V, 1933; Vol. VI, 1934; Vol. VII, The University of Toronto Press, Toronto, 1950. *De Bary, a.: Comparative Morphology and Biology of the Fungi, Mycetozoa and Bacteria (trans. H. E. F. Garnsey), Oxford University Press, New York, 1887. Dodge, B. O. : Some problems in the genetics of the fungi. Science 90 : 379-385, 1939. *DuBos, R. J.: Louis Pasteur, Free Lance of Science, Little, Bro^Ti & Company, Boston, 1950. Kauffman, C. H.: Klebs' theory of the control of developmental processes in organisms, and its application to fungi, Proc. Intern. Congr. Plant Sci. 2 : 1603- 1611, 1929. Klebs, G.: Zur Physiologic der Fortpflanzung einiger Pilze. III. Allgemeine Betrachtungen. Jahrb. wiss. Botan. 35 : 80-203, 1900. Orton, C. R.: Leon Hatchig Leonian. Phytopathology 36: 241-244, 1946. Pasteur, L.: Oeuvres de Pasteur, reunies par Pastevur Vallery-Radot, 7 vols., Masson et Cie, Paris, 1933-1939. ScHOPFER, W. H.: Plants and Vitamins, Chronica Botanica Co., Waltham, 1943. CHAPTER 2 CULTURE MEDIA Before discussing the nutrition of the fungi in detail, it will be helpful to consider the basic problems involved. For many purposes a knowledge of the nutrition of the fungi is necessary for culturing them in the labora- tory or in industry. Like all living organisms the fungi must obtain from their environment the materials needed for the synthesis of protoplasm and other cellular constituents. Directly or indirectly, the fungi as well as animals and most bacteria are dependent upon green plants for "food" and energy. Not all natui'al substrates are equally suitable for all fungi. In nature, the saprophytes are more widely distributed than the parasites, which are usually restricted to the range of their hosts. Many of the substances upon which the fungi grow in nature are chemically complex, and some, such as cellulose, starch, and proteins are insoluble or are only colloidally soluble. Before such compounds can be utilized, they must be changed into low-molecular-weight compounds which are soluble in water. This "digestion" is accomplished by means of enzymes which are excreted by the fungi. This is analogous to digestion in animals, which is also an enzymatic process. The complete utilization of a natural substrate is frequently due to the combined action of a succession of microorganisms. More than one organism may act at the same time, and often this simul- taneous action is more effective than that of a single organism. One may ask. Do the fungi simply incorporate within their own proto- plasm the suitable elements and compounds found in the medium, or do they transform the compounds of the medium before building their own structures? Apparently the fungi do both. The essential elements such as potassium and magnesium are taken up as ions, although these ele- ments may be in the state of chemical combination in the substrate and also in the fungus cells. Certain organic compounds, such as the vita- mins, are undoubtedly absorbed as such from the medium by vitamin- deficient fungi; otherwise, these fungi would derive no benefit from them. The same statement is true for other necessary compounds which the various fungi are unable to synthesize. By far the greater part of the compounds utilized by the fungi are modified or changed either before or after they are taken into the cells. Outside the fungus cells, these changes are largely in the direction of simplifying the molecular structure of compounds used. Within the fungus cells some of the metabolite molecules are oxidized to carbon 8 CULTURE MEDIA 9 dioxide and water or to intermediate products. By this process the fungus obtains the chemical energy which it requires for the processes of synthesis. KINDS OF MEDIA No one knows when man began to cultivate fungi, but certainly it was many thousands of years ago. This cultivation was no doubt uninten- tional at first and was later developed into an art, in connection with the preparation of foodstuffs and beverages. The use of leaven (yeast) extends back to the beginning of agriculture. The yeast culture was preserved in a piece of dough which in turn was added to the next batch, much as buckwheat batter is prepared today. In the Orient, species of Mucor and Aspergillus have been used from the dawn of civilization in preparing food from rice and soybeans. Brewers used yeast many cen- turies before it was learned that yeast is a living organism. On the other hand, the science of growing fungi in pure culture is fairly recent. Natural media. It was quite natural that, when mycologists and others began to cultivate fungi in the laboratory, they should turn to natural materials as media. A natural medium is one which is composed entirely of complex natural materials of unknown composition. Among the natural substances so used are the following: plant parts, malt, yeast, peptone, manure, bread, wort, fruit, and vegetables. Many of these substances are used in the form of extracts, infusions, or decoctions. The very diversity of these natural media is strong testimony to the fact that different species have different nutritional requirements. Brefeld (1881) was among the first to grow fungi in pure culture, and many of his tech- niques are in use today. Since his interest in cultivating the fungi was largely for the purpose of observing their development, it was necessary for him to select suitable media. He found two natural media to be of great utility: a decoction prepared from dried plums or raisins and a manure extract. This latter medium he considered "als Universal- nahrlosung flir Pilzculturen." This medium is still used in some labora- tories. Natural media have many advantages. They are cheap and easy to prepare. In many instances it is necessary only to add water to the base material and autoclave. More important yet is the fact that many fungi grow well upon a wide variety of natural media. Certain of the more fastidious fungi have never been cultivated in the laboratory. These obligate parasites live only upon or ^^^thin the living tissues of their hosts. Puccinia graminis tritici lives only on wheat, some species of grasses, and some species of barberry. These host plants when killed will no longer support growth of this fungus. However, many species, which in the past were considered to be obligate parasites, have since been cultured on nonliving media. 10 PHYSIOLOGY OF THE FVSGI Semisynthetic media. A semisynthetic medium is one Avhich is com- posed in part of natural materials of unknown composition. Such media are made by adding compounds of known composition to one or more natural materials. The widely used potato-glucose (dextrose) medium is an example of this type. The addition of agar to an otherwise synthetic medium introduces a natural material of unknown composition. Media Avhich contain agar cannot be classed strictly as synthetic media. Semi- synthetic media may be used for many types of physiological investigations. The composition of a given natural or semisynthetic medium is not constant. Potato-glucose medium may vary greatly in composition depending upon whether or not the potatoes were peeled and upon the variety and age of the potatoes used. Neuberger and Sanger (1942) found a twofold difference in the amide nitrogen (asparagine and gluta- mine) among varieties. In this laboratory we have found that the amount of potato pulp Avhich is allowed to enter this medium exerts a marked influence upon growth and reproduction of certain fungi. These differences, which may seem minor, are great enough to make comparisons between work done in different laboratories difficult. Synthetic media. As the term is used in this book, a synthetic medium is one of known composition and concentration. It does not mean that every compound used is a product of the chemist's art. Some of the constituents, such as the sugars, may be of natural origin. The important condition is that the compounds used be pure, and this is difficult to attain in practice. "Chemically pure" compounds are usually far from being pure, as a glance at the labels will show. The ideal of using pure compounds is seldom realized, but the closer it is approached, the more we shall learn about the nutrition of the fungi. Natural media and most semisynthetic media are of limited usefulness in studying nutrition of the fungi. The chief value of synthetic media is for nutritional studies. However, growth and reproduction are fre- quently poorer on a synthetic medium than on one containing some natural material. For example, Aspergillus niger grew well in a synthetic medium composed of sucrose, ammonium nitrate, magnesium sulfate, and dipotassium hydrogen phosphate (Steinberg, 1939). In addition to these major constituents, iron, zinc, copper, manganese, molybdenum, and gallium salts were present. Extraordinary care was taken in prepar- ing the medium. The concentration of every constituent was so balanced that a decrease in concentration of any constituent resulted in diminished growth. Growth and sporulation were excellent upon this medium. When 20 mg. per liter of either peptone or yeast extract was added, the rate of growth was greatly increased and the time required for sporulation was decreased. The small amount of yeast extract or peptone used could have added only an insignificant amount of material from which CULTURE MEDIA ii the fungus could synthesize protoplasm or derive energy. Steinberg's synthetic medium was adequate but not optimum for most rapid growth and sporulation. We may suppose that the yeast extract and peptone contained compounds the synthesis of which constituted a limiting effect upon the rate of growth and sporulation. Synthetic media may be simple or complex but must contain the essen- tial elements in utilizable form. Brefeld (1881) gave the following direc- tions for preparing a synthetic medium: Add cigar ashes dissolved in nitric or citric acid to a solution containing a soluble carbohydrate, such as glucose, and an ammonium salt. The amount of ashes was not speci- fied. The first vsynthetic medium was devised by Raulin (1869). Table 1. Composition of the First Synthetic Medium for CuLTrvATiNG Fungi (Raulin, Ann. sci. nat., Ser. V, 11, 1869.) Ammonium nitrate 4.0 g. Ammonium phosphate 0.6 g Magnesium carbonate 0.4 g Potassium carbonate 0.6 g Ammonium sulfate 0 . 25 g Zinc sulfate 0 . 07 g, Iron sulfate 0 . 07 g Potassium silicate 0 . 07 g, Sucrose 70 g, Tartaric acid 4 g Water 1,500 ml. However, not enough information is given in Table 1 for the duplication of this medium. Which ammonium phosphate, (NH4)H2P04 or (NH4)2- HPO4, was used by Raulin in the original work? Which zinc sulfate, ZnS04-7H20 or ZnS04-H20, was used? Was the iron sulfate FeS04, FeS04-7H20, or re2(S04) 3? Did he use D-tartaric, L-tartaric, DL-tartaric, or weso-tartaric acid? These questions are asked for the purpose of emphasizing the need for exactness in reporting the composition of media used in experimental work. These uncertainties creep into the literature through ignorance or carelessness, or both. Nor are these ambiguities to be found only in the older literature, for they are present in papers pub- lished only yesterday. Either the specific name or the formula, or both, should be stated. If it is stated that dipotassium phosphate, K2HPO4, was used, the reader is certain of the identity of the compound. Potas- sium phosphate may designate at least five distinct chemical compounds. NATURAL VERSUS SYNTHETIC MEDIA In addition to the fact already noted that the composition of natural media is unknown, natural and synthetic media differ in two further respects. Natural media are more complex; i.e., they contain more 12 PHYSIOLOGY OF THE FUNGI chemical compound.s than synthetic media. They also contain com pounds ordinarily not present in synthetic media. Specific metabolites. Only certain chemical compounds are utilized by fungi, but not all fungi are able to utilize the same compounds. Any compound utilized by a fungus is called a metabolite. Some fungi are unable to synthesize certain essential metabolites and are said to be "deficient" for the specific metabolites they are unable to synthesize. In order to cultivate such deficient fungi, these metabolites must be pres- ent in the medium. Natural media usually contain these metabolites. If a fungus grows upon a natural medium and fails to grow upon a variety of simple synthetic media, it may be suspected that specific metabolites are involved in its nutrition. The following example will illustrate the role of specific metabolites in fungus nutrition. Fellows (1936) investigated the ability of Ophiobolus grayninis to utilize different nitrogen compounds for growth. A sucrose- mineral salts solution was used as the basal medium to which various nitrogen sources were added. Only complex nitrogen sources such as egg albumen, peptone, casein, and nucleic acid allowed growth. Under the conditions used no growth resulted when ammonium compounds, nitrates, nitrites, and amino acids were tested. 0. graminis in the presence of egg albumen utilized glucose, maltose, lactose, fructose, xylose, starch, and dextrin, in addition to sucrose. From these experimental results it was concluded that 0. graminis requires a complex source of nitrogen for growth. Later, White (1941) found that this fungus requires two specific metabolites, thiamine and biotin. When these vitamins were added to synthetic media containing simple nitrogen sources (sodium nitrate, ammonium nitrate, asparagine, or glycine), good growth was obtained. Thus, it appears obvious that 0. graminis does not require a complex nitrogen source, but that it is unable to synthesize two specific chemical compounds. These papers illustrate the fact that fungus physiology is a young and developing science. Much of the early work needs reevalua- tion in the light of recent discoveries. A student should strive to develop a critical attitude toward the work of others, but he should be no less critical with regard to his own work. The evaluation of experimental results depends upon the conditions under which the work was done, and among these conditions the medium used is of first importance. Complexity of media. It is a common experience to find that a trace of some crude natural product stimulates the rate of growth and sporulation of a fungus. This stimulation frequently occurs with fungi which grow well on synthetic media and which are not deficient for vitamins or amino acids. It appears that the complexity of natural media offers a clue to understanding this stimulatory effect. If a fungus is grown upon a simple CULTURE MEDIA 13 synthetic medium which has only one source of carbon and one source of nitrogen, it must synthesize many complex chemical compounds from constituents present in the medium. It may be suspected that these biochemical syntheses are slowed up under these conditions. When a mixture of many carbon and nitrogen sources is present, the fungus may function more efficiently, because the biochemical syntheses are easier since some of the intermediates are furnished. These speculations receive some support from evidence to be presented in Chaps. 6 and 7_ CHOICE AND PREPARATION OF MEDIA Considerable care is needed in the selection of a suitable medium. A medium may be excellent for growth and unsuitable for reproduction or the production of an antibiotic. The method of preparation may influ- ence the composition of a medium in unsuspected ways. Choice of media. In selecting a medium the purpose for which it is to be used should be kept in view. For many purposes a natural medium is the one of choice. This is especially true for routine maintenance of cultures, for isolations, and for preliminary investigations. The composi- tion of natural media may be varied by choosing different substrates. Frequentl}^, a combination of natural products may be used to advantage, e.g., malt and yeast extracts. In addition, these natural substrates may be fortified with one or more pure chemical compounds. The constitu- ents of natural media are fixed by the substances used, but the amounts used may be changed at will. More judgment enters into the selection of synthetic media. The essentials of a synthetic medium may be stated as follows : sources of car- bon and nitrogen in utilizable forms; phosphate and sulfate ions; the metallic ions potassium, magnesium, iron, zinc, manganese, and others which are usually present as impurities in the chemicals used. These are the essential elements and will be considered at length in later chapters. Most fungi utilize glucose, so this sugar is frequently used as the carbon source. More fungi utilize nitrogen in organic combinations than in inorganic compounds. The question of specificity enters into the choice of the carbon and nitrogen sources, and this can be determined only by experiment. In order to cultivate deficient fungi on synthetic media, the specific metabolites for which the fungi are deficient must be added. Since synthetic media are used to study nutrition, the development of a suitable synthetic medium for a specific fungus may require considerable investigation. In our laboratory we commonly first use a glucose-casein hydrolysate medium containing the essential inorganic elements. This medium has been very useful in vitamin studies. Its composition is given in Chap. 10. 14 PHYSIOLOGY OF THE FUNGI Solid versus liquid media. Both solid and liquid media are used in cultivating fungi. Media solidified with agar, or semisolid substrates such as corn meal, offer many advantages in that the culture vessels can be freely handled without disturbing the fungus. This feature is particu- larly valuable when one wishes to follow the development of a fungus. Microscopic examination is facilitated, and contaminants are more easily detected. Single-spore isolations can be made more easily from solid media. Agar media are used to maintain stock cultures and are recom- mended for many preliminary experiments. Frau Hesse (Kitchens and Leikind, 1939) introduced the use of agar into microbiological procedures in 1881. Agar, which is obtained from various marine red algae, is a complex polysaccharide sulfate ester (Pigman and Goepp, 1948). It forms colloidal solutions at elevated temperatures and sets to a gel at temperatures around 45°C. On acid hydrolysis both D-galactose and its enantiomorph, L-galactose, as well as sulfuric acid, are formed. Agar must exist in the form of a salt (Ca, Mg, Na, K, etc.) to form a gel. Agar introduces physiologically active elements into media. It may contain significant amounts of zinc (Leonian and Lilly, 1940) and other micro essential elements. Mulder (1940) found that magnesium could be efficiently removed from agar by repeated soakings in 10 per cent sodium chloride solution, followed by washing with distilled w^ater until the filtrate was free from chloride ion. Agar also contains growth factors such as thiamine (Day, 1942) (see Fig. 1). Many fungi make some growth on water agar, which indi- cates that agar or the ''impurities" contained in it are utilized by fungi. Robbins (1939) found that leaching agar with 5 per cent aqueous pyridine removed many of the physiologically active compounds. Liquid media should be used for precise investigations where it is desired to control as many variables as possible. The composition of the medium may be controlled and the amounts used measured accurately. Cultures may be aerated by shaking or by blowing sterile air through the media. Weighing the mycelium is facilitated. When it is desired to study the metabolic by-products of fungus metabolism (except gaseous products), it is almost necessary to use liquid media. Isolation of by-products is less complicated when liquid media are used. Studies of various metabolite deficiencies and many microbiological assays (Chap. 10) almost always require the use of liquid media. The choice between the use of solid or liquid media should be made on the basis of the known advantages and disadvantages of both and with regard to the purpose of the problem under investigation. Designating media. It is common to find references to a medium by the name of the investigator who first used it. These names have served as convenient abbreviations and commemorate the pioneers in the art of CULTURE MEDIA 15 cultivating fungi. Some of these names are Blakeslee, Uschinsky, Coons, Czapek, Leonian, Sabouraud, Richard, Thaxter, Shear, Raulin. From a historical standpoint this practice has much to recommend it. However, this usage has many disadvantages. These distinguished names give no clue to the composition of these media. The original formulas have in many instances been changed. Some of these modifications have received A B Fig. 1. Growth of Phycomyces blakesleeanus on vitamin-free liquid medium solidified with two different brands of agar. Growth in A indicates relatively high content of thiamine of this agar. The trace of growth in B shows that this agar is relatively free of thiamine. hyphenated names: e.g., Czapek-Dox. Frequently the originator of a medium modified it from time to time. This introduces a further uncer- tainty as to its composition. In our opinion the use of personal names to designate media should be abandoned. It is much more helpful to designate media by descriptive titles than by names which tell nothing of the composition. The carbon and nitrogen sources are important con- stituents of every medium. Thus, sucrose-nitrate medium, glucose- asparagine medium, or malt extract-yeast extract medium are preferred to Czapek's medium, Schopfer's medium, or Leonian's medium. These descriptive terms afford valuable information that personal names do not. Even when the reader is familiar with the composition of a named medium, 16 PHYSIOLOGY OF THE FUNGI there is a tendency to fail to associate experimental results with the composition. Effect of autoclaving. Media are commonly and effectively sterilized by autoclaving. It should be noted, however, that such high tempera- tures may cause destruction or alteration of some constituents in the media. These changes are not serious for many uses; at least media pre- pared in this way are satisfactory. Sugars are among the substances most easily altered by autoclaving. The extent of decomposition depends upon the specific sugar used, the other constituents of the medium, and the time of autoclaving. It is desirable to adopt a uniform schedule for autoclaving media. An increase in the amount of caramelization occurs as the time of heating is increased. Maillard (1912) showed that a brown color results when reducing sugars (glucose, fi-uctose, etc.) are autoclaved with amino acids. Hill and Patton (1947) have shown that growth of Streptococcus faecalis is reduced when tryptophane is autoclaved with sugars. Margolin (1942) found that no one method of sterilization resulted in best growth for all of the 14 species tested. Phythophthora erythroseptica made three times the amount of growth on glucose sterilized by filtration as when the entire medium was autoclaved. Syncephalastrum racemosum, however, made more growth on autoclaved than on sterile- filtered glucose (Table 2). The organisms most sensitive to heated glucose appear to be various species of Cytophaga, which failed to grow on glucose which had been heated to 50°C. (Stanier, 1942). These organisms utilized glucose which had been sterilized by filtration. Phos- phates, a universal constituent of media, are active in converting glucose into ketoses and other products (Englis and Hanahan, 1945) during autoclaving. Complex sugars and polysaccharides undergo some hydrolysis during autoclaving. The amount of hydrolysis is dependent upon the carbo- hydrate, the time and temperature of autoclaving, and the pH of the medium. Sucrose, when autoclaved in acidic media, may undergo suffi- cient hydrolysis to support some growth of species unable to utilize sucrose. This possibility must be guarded against in experiments on the availability of complex sugars. Other substances used in media may be destroyed during autoclaving. To minimize or avoid such effects, heat-sensitive substances may be auto- claved separately, or they may be sterilized using special bacteriological filters. The Berkefeld and Chamberland filters are less used than formerly, while at present Seitz and fritted-glass filters are widely used. Fritted-glass filters are best for most purposes, inasmuch as the asbestos pad used in the Seitz filter may adsorb active compounds. All methods of sterilization w^hich depend upon filtration are slow and can be used only with liquid media. Various volatile chemical sterilization agents such as CULTURE MEDIA 17 alcohol and acetone have been used. Hansen and Snyder (1947) have recommended the use of propylene oxide for the sterilization of plant parts used for culture media. Frequently a seemingly insignificant change in the method of preparing a medium may result in significant changes in the composition of the medium, which in turn may be reflected in the behavior of the organisms grown upon it. Even the volume of medium in culture vessels affects the amount of decomposition during autoclaving. Cotton plugs may introduce hnt into the medium. Less refined grades of cotton release a volatile substance which affects the Table 2. The Effect of Different Methods of Sterilizing Glucose upon THE Growth of Sex Fungi, at 25°C. Growth reported as milligrams of dry mycelium. The entire medium, containing a mixture of amino acids, was autoclaved in the control experiment. In the other experiments the glucose was sterilized by either Seitz filtration or treatment with acetone and added aseptically to the remainder of the sterile medium. (Margolin, thesis. West Virginia University, 1942.) Species Days of incuba- tion Control, entire medium autoclaved Glucose sterilized by filtration Glucose sterilized by treating with acetone Phycomyces blakesleeanus Rhizopus suinus 7 6 5 12 15 15 130 122 103 79 84 142 140 123 68 241 88 147 132 115 80 192 85 103 Syncephalastrum racemosxun Phytophthora erythroseptica Diplodia macrospora Phytophthora cadonim germination of some spores {Phycomyces blakesleeanus, Robbins and Schmitt, 1945). Paper or aluminium caps may be used to replace cotton plugs. Residual soap films on improperly rinsed glassware may cause trouble in some cases. Preparation of media. Directions for the preparation of specific media are given at the end of the text in the section Suggested Laboratory Exercises. Additional details concerning various media are to be found in Riker and Riker (1936) and Rawlins (1933). WAYS OF EXPRESSING CONCENTRATION Concentrations are frequently expressed in the literature as percentages. Unless the basis for calculating these values is given, percentage is an ambiguous way of reporting concentration. Buchanan and Fulmer (1928) have pointed out that there are six ways of calculating the percent- age composition of a solution. A 10 per cent sulfuric acid solution may represent six different concentrations. For any precise work it is best to 18 PHYSIOLOGY OF THE FUNGI avoid the use of percentages, but for routine work, where the composition of media is of less importance, the use of percentages may be allowed. Before the same medium can be prepared repeatedly, it is necessary to know what constituents are used and the amount of each. Two general methods are used for reporting the composition of media. Either the weights of the constituents and the volume of water used are given, or the weights of the constituents are given and the medium made up to a definite volume. The first method is in common use; its sim- plicity conceals its disadvantages. The volume of a medium prepared by this method is never the same as the volume of water used. It is neces- sary to measure the volume of the medium after preparation in order to calculate the amount of any constituent in an alicjuot. The method of choice in accurate work is to weigh the constituents and make the medium up to a given volume. The amovmt of any constituent in any volume of medium may then be calculated. If a liter of medium contains 25 g. of sucrose, and 25-ml lots are dispensed, each lot contains ^/iooo X 25, or 0.625 g. of sucrose. Direct units. The units of volume most used are the liter (1.) and the milliliter (ml.). A cubic centimeter (cc.) is nearly, but not exactly, equivalent to a milliliter. Its use should be discouraged. The formulas for media are usually given on the basis of a liter. This practice is to be encouraged, as the liter is a convenient volume in preparing media. The weights of solid constituents should be reported as grams (g.) or decimal divisions thereof. The most commonly used decimal fractions of the gram are the milligram (mg.), the microgram (jug), and the millimicrogram (m/ig), each of which is one-thousandth of the preceding weight. Since it is easy to make mistakes in reading small decimals, it is recommended that no decimals smaller than 0.1 be used. The use of 12 mg. is preferable to 0.012 g., although both mean exactly the same. It is easier to read 5 jug than 0.000005 g. One milligram of a substance in a Uter of solution equals one part per million (p. p.m.). Each milliliter of such a solution wiU contain 1 fxg of the substance. Similarly, a microgram of a substance in a liter of solution is present as one part per billion. The microgram has also been called the gamma (7), but this usage should be abandoned inasmuch as gamma is not a regular prefix used in the metric system. The necessity of using such small units of weight arises from the physio- logical activity of certain compounds and elements. For example, a concentration of 1 mg. of biotin in a liter of medium is a relatively enor- mous concentration. Derived units. Derived units must be used in comparing the effect of compounds which have different molecular weights. Among these derived units the mole is the most useful. A mole is the molecular weight of a chemical compoimd expressed in grams. A mole of glucose is 180 g., \^ K^ ±^ J. \^ X\/X-J 1.WX JUt X-^X iX. while a mole of sucrose is 342 g. A liter of solution containing one mole of a compound is said to be one molar (il/). Equimolar solutions contain the same number of molecules. In problems in physiology, such as osmotic pressure, which have to do with the numbers of molecules it is necessary to use this way of expressing concentration. If it is desired to compare the effect of the osmotic pressure due to glucose and sucrose, the concentration must be expressed in terms of molar strengths, for the osmotic pressure is a function of the number of molecules of solute in a solution. If it is the purpose to compare the effect of glucose and sucrose on the amount of growth of a fungus, this method of expressing concen- trations should not be used. Media of equal molarity with respect to sucrose and glucose do not contain the same amount of carbon. The first contains twice as much carbon as the second. Just as a milligram is one-thousandth of a gram, a millimole is one-thousandth of a mole. The meaning of micromole and millimicromole should be obvious. If the weight of a compound is given in grams, this datum may be con- verted into moles. If a medium contains 50 g. of glucose per liter, the glucose concentration may be expressed as 50/180 or 5/18ilf. Con- versely, if the concentration of sucrose in a medium is stated to be 0.15ilf, the weight of sucrose is 0.15 X 342 or 51.3 g. per liter. These conversions imply that the molecular weight is known or can be calculated. In pre- paratory work compounds are weighed on a balance as grams, not as moles, and unless the interpretation of the results demands conversion to moles, it is better to record the weights than to convert these data to derived units. The mole and molar solutions are particularly useful in dealing with non-ionizing compounds. Another derived unit, the equivalent, is frequently used to express the concentration of ionized compounds. An equivalent is the atomic weight of an ion expressed in grams divided by the valence of the ion. If an ion is composed of more than one atom, the ion weight is computed by adding together the atomic weights. It is important to remember that, if an element has more than one valence, the equivalent weight depends upon the valence. An equivalent of ferrous (Fe++) ion is 55.8/2 or 27.9 g., while an equivalent of ferric (Fe+++) ion is 55.8/3 or 18.6 g. A normal solution (A^) is one which contains one equivalent in a liter of solution. In dealing with small amounts it is convenient to use milliequivalents or microequivalents. In preparing a series of media for the purpose of comparing the growth of a fungus on different nitrogen sources, the nitrogen content of the media should be equal. If urea, CO(NH2)2, and aspartic acid, HOOC — CH2 — CH(NH2) — COOH, are used, it is obvious that different weights of these nitrogen sources must be used if the media are to contain equal amounts of nitrogen. Whenever media are modified by replacing one compound by 1 20 PHYSIOLOGY OF THE FUNGI another, it should be done in such a way that the same amount of the essential element is present in all the media. If this is not done, the basis upon which the replacement was made should be stated. If 25 g. of glucose, C6H12OC is replaced by 25 g. of sucrose, C12H22O11, it should be realized that the carbon contents of the two media are different. It is frequently difficult or impossible to find out from some papers in the literature how substitutions in the media were made. Table 3. A Compakison of Two Synthetic Media upon the Basis of Amounts OF Essential Elements and Compounds Present in One Liter Both media were made with double-distilled water. Glucose-asparagine * Sucrose-ammonium nitrate f Element or compound Unit of meas. Source Unit of meas. Source c G. 4.0 0.427 0.049 0.065 0.287 0.228 Mg. 0.2 0.2 0.1 ^g 100 5 D-Glucose, 10 g. L-Asparagine, 2 g. MgS04-7H.,0, 0.5 g. MgS04-7H.20, 0.5 g. KH2PO4, 1.0 g. KH,P04, 1.0 g. As sulfate As sulfate As sulfate G. 21.4 0.720 0.025 0.032 0.125 0.062 Mg. 0.3 0.3 0.075 0.075 0.02 0.02 Sucrose, 50 g. N NH4NO3, 2.06 g. Mg MgS04-7HoO, 0.25 g. S MgS04-7H,0, 0.25 g. K K2HPO4, 0.35 g. P K2HPO4, 0.35 g. Fe As chloride Zn As chloride Mn Cu As chloride As chloride Mo As chloride Ga As chloride Thiamine hydrochloride Biotin * Medium 5, Suggested Laboratory Exercises, t Steinberg, 1941. Finally, it should be noted that the common practice of using one com- pound as the source of two essential elements does not permit perfect freedom in adjusting the composition of a medium. If magnesium sulfate heptahydrate is used to supply both magnesium and sulfur, it is obvious that the ratio Mg/S is fixed. If it is desired to vary the amounts of magnesium and sulfur independently, it is necessary to use different com- pounds of magnesium and sulfur; e.g., magnesium chloride and sodium sulfate. This practice introduces other elements into the medium. CULTURE MEDIA 21 COMPARISON OF MEDIA Media differ only in constituents and amounts used. It is desirable to be able to compare media in some uniform way. To do this, it is neces- sary to know not only the amounts of the elements present, but also the compounds in which these elements occur. A comparison of two syn- thetic media is given in Table 3. From Table 3 it will be noted that these media contain the same essen- tial elements. Copper, molybdenum, and gallium do not appear in the composition of the glucose-asparagine medium, but it should not be con- cluded that these elements were not present, since only c.p. chemicals were used to prepare this medium. Stout and Arnon (1939) note that a distinction must be made between ordinary chemical purity and biological purity. This will be considered in detail in Chap. 5. The two features which make these media quite distinct are the different sources of carbon and nitrogen used and the addition of two vitamins to the glucose- asparagine medium. The latter medium is suitable for the growth of more species of fungi than is the sucrose-ammonium nitrate medium. SUMMARY Fungi secure food and energy from the substrates upon which they live in nature. In order to culture fungi in the laboratory, it is necessary to furnish in the medium those essential elements and compounds they require for the synthesis of their cell constituents and for the operation of their life processes. The synthetic abilities of fungi differ. Some fungi are unable to s5Tithesize certain key compoimds that they require and must obtain them from the medium upon which they grow. All the fungi require much the same essential elements but differ widely in their ability to utilize compounds in which these elements occur. There is no uni- versal natural substrate or artificial medium upon which all fungi will grow. On the basis of composition there are three general types of media: natural media, which are composed entirely of natural products; semi- synthetic media, which are composed in part of natural substances; and synthetic media, which are of kno^\^^ composition. Natural media are most useful for routine work, while synthetic media and, to a limited extent, semisynthetic media are used to investigate the nutritional requirements of the fungi. Media differ only with respect to constituents and concentrations. The compounds and the amounts used in preparing a medium must be specified exactly. Media should be designated by naming the carbon and nitrogen sources used, e.g., glucose-asparagine medium. The use 22 PHYSIOLOGY OF THE FUNGI of proper names to designate the composition of a medium should be avoided. The selection of a suitable medium depends upon the fungus under study and the purpose of the experiment. Not all media are equally suitable for all fungi, nor is one medium suitable for a complete physio- logical study of one fungus. REFERENCES Brefeld, O.: Botanische Untersuchungen liber Schimmelpilze, Heft, IV Verlag Arthur Felix, Leipzig, 1881. Buchanan, R. E., and E. I. Fulmer: Physiology and Biochemistry of Bacteria, Vol. I, The Williams & Wilkins Company, Baltimore, 1928. Day, D.: Thiamin content of agar. Bull. Torrey Botan. Club 69: 11-20, 1942. Englis, D. T., and D. Hanahan: Changes in autoclaved glucose, Jour. Am. Chevi. Soc. 67 : 51-54, 1945. Fellows, H.: Nitrogen utilization by Ophiobolus graminis, Jour. Agr. Research 53: 765-769, 1936. *Hansen, H. N., and W. C. Snyder: Gaseous sterilization of biological materials for use as culture media, Phytopathology 37: 369-371, 1947. Hill, E. G., and A. R. Patton: The Maillard reaction in microbiological assay. Science 105 : 481-482, 1947. HiTCHENS, A. P., and M. C. Leikind: The introduction of agar-agar into bacteri- ology. Jour. Bad. 37: 485-493, 1939. Leonian, L. H., and V. G. Lilly: Studies on the nutrition of fungi. IV. Factors influencing the growth of some thiamin-requiring fungi. Am. Jour. Botany 27: 18-26, 1940. Maillard, L. C: Action des acides amines sur les sucres; formation des melanoldines par voie methodique, Conipt. rend. acad. sci. 154: 66-68, 1912. Margolin, A. S.: The effect of various carbohydrates upon the growth of some fungi, thesis. West Virginia University, 1942. Mulder, E. G. : On the use of micro-organisms in measuring a deficiency of copper, magnesium and molybdenum in soils, Antonie van Leeuwenhoek 6 : 99-109, 1939-1940. Neuberger, a., and F. Sanger: The nitrogen of the potato, Biochem. Jour. 36: 662-671, 1942. Pigman, W. W., and R. M. Goepp, Jr.: Chemistry of the Carbohydrates, Academic Press, Inc., New York, 1948. Raulin, J.: Etudes chimiques sur la v^g6tation, Ann. sci. nat., Ser. V, 11: 93-229, 1869. Rawlins, T. E.: Phytopathological and Botanical Research Methods, John Wiley & Sons, Inc., New York, 1933. RiKER, A. J., and R. S. Riker: Introduction to Research on Plant Diseases, John S. Swift Co., St. Louis, 1936. *RoBBiNS, W. J.: Growth substances in agar. Am. Jour. Botany 26: 772-778, 1939. Robbins, W. J., and M. B. Schmitt: Effect of cotton on the germination of Phyco- myces spores, Bull. Torrey Botan. Club 72 : 76-85, 1945. Stanier, R. Y. : The Cytophaga group: a contribution to the biology of myxobacteria, Bact. Revs. 6 : 143-196, 1942. Steinberg, R. A.: Relation of carbon nutrition to trace-element and accessory requirements of Aspergillus niger, Jour. Agr. Research 59: 749-763, 1939. CULTURE MEDIA 23 Steinberg, R. A.: Sulfur and trace element nutrition of Aspergillus niger, Jour. Agr. Research 63: 109-127, 1941. Stout, P. R., and D. I. Arnon: Experimental methods for the study of the role of copper, manganese, and zinc in the nutrition of higher plants, Am. Jour. Botany 26: 144-149, 1939. *■ White, N. H.: Physiological studies of the fungus Ophiobolus graminis Sacc, Jour. Council Sci. Ind. Research 14: 137-146, 1941. CHAPTER 3 GROWTH Growth may be considered either as an increase in cell number or as an increase in mass. Usually both these processes are concurrent in the phenomenon called growth. To a limited degree, fungus cells may divide and form new cells without an increase in mass. A spore may germinate in distilled water and give rise to a germ tube, but in the absence of nutrients this process soon stops. A few cell divisions exhaust the reserve material originally present in the spore, and growth soon ceases unless these new cells obtain nutrients from the external environ- ment. Under certain conditions fungus cells may increase their store of reserve materials, and thus their mass, without an increase in cell number, but this process is also limited. Growth, excluding the limited meanings given above, involves an increase in both the number and the mass of cells. This definition of growth neither ''explains" the processes involved nor indicates their complexity. Rahn (1932) has expressed doubt that we will ever fully understand the process of growing. A yeast cell which buds and produces a daughter cell illustrates one of the striking features of growth : growth involves duplication. From a dozen or so simple chemi- cal substances present in the medium the parent cell synthesizes at least a portion of the protoplasm of the daughter cell. The daughter cell has the same genetic constitution as the parent cell, and thus a duplication of genes is a feature of cell multiplication. The compounds which comprise protoplasm, enzymes, genes, and other substances are extraordinarily complex. Our meager knowledge concerning the chemical architecture of these substances only confirms this view. In the synthesis of such compounds we may assume that the chemical reactions which produce them are perfectly timed and coordinated, for no series of uncorrelated reactions could produce such compounds. The growth processes of the filamentous fungi are still more complex than those of yeast, because of greater differentiation in structure. In those species of fungi which produce aerial mycelium these parts are nourished through the mycelium in contact with the medium. This involves translocation of nutrients over considerable distances. This is especially true of sporangiophores and aerial fruit bodies. The develop- ment of fruiting structures and spores is growth, in that the formation of new cells is involved. The formation of fruit bodies in many species 24 GROWTH 25 takes place at the expense of reserve materials and protoplasm formed by and stored in the vegetative mycelium. PHASES OF GROWTH Growth in the fungi, as in other organisms, follows a definite pattern. The way this development takes place depends upon the species and the environmental and nutritional conditions. In the present discussion, it will be assumed that the external conditions are favorable and that growth takes place in a limited volume of medium. Unicellular organisms. The bacteriologists have long been interested in the mathematical analysis of the phenomenon of growth. The student is referred to Buchanan and Fulmer (1928) and to Rahn (1932, 1939) for further information on this subject. Among the fungi, the yeasts have somewhat the same type of development as the bacteria. Since bacteria multiply by fission and the yeasts (except Schizosaccharomyces) by budding, we cannot expect the growth pattern of yeasts to fit exactly the same formulas which have been developed for bacteria. But, in a general way, yeasts follow closely the phases of growth shown by bacteria. These phases of growth are as follows: (1) Stationary phase. When cells are inoculated into a medium, there is a period of time following inocu- lation when there appears to be no change in number. The stationary phase may be long or short depending upon the age and vigor of the inoculum, the medium, and other factors. (2) Phase of accelerated growth. Not until cell division is established and new protoplasm is being formed from the constituents of the medium may growth be considered as begun. This phase is characterized by an increase in the rate of cell division, i.e., the generation time is decreasing. (3) Exponential or logarithmic phase. This phase is clearly defined for bacteria and approached by yeasts. It is characterized by a constant generation time. If the logarithms of the cell numbers are plotted against time, the curve is a straight line. (4) Phase of declining acceleration. As the nutrients become exhausted, or as toxic by-products accumulate, the average generation time increases. A combination of these and other factors results in a lessened rate of growth. If fresh medium were continuously supplied and toxic by-prod- ucts removed, it is possible that this phase would never be attained. (5) Maximum stationary phase. This marks the attainment of maximum weight, or numbers of living cells. It is quite likely that the death of old cells is balanced by new growth. The duration of this phase is dependent upon the organism and upon the composition of the medium at this time. (6) Phase of decline or autolysis. Sooner or later, following attainment of maximum development, autolysis sets in. As the cells die, the cellular enzymes begin to digest the various cell constituents. Only the more resistant portions of the cell remain. Microscopic examination at this 26 PHYSIOLOGY OF THE FUNGI time reveals that many cells are devoid of protoplasm. It is quite possi- ble that some of the materials released by autolysis are used by the remaining living cells. Filamentous fungi. With exception of the third phase of growth dis- cussed above, the filamentous fungi follow the same order of development as the yeasts. The most obvious difference between the filamentous fungi and unicellular organisms is the failure to attain an exponential rate of growth. Usually, the exponential phase is replaced by a more or less linear phase of growth. Emerson (1950) found a straight-line relation between the cube root of the weight of mycelium produced by Neurospora crassa grown in nonagitated liquid medium and the time of incubation. This relation held for three surface-volume ratios. A comparison of the linear, logarithmic, and cube-root growth curves indicates that this fungus has a cube-root phase of growth during the interval when the linear graph is concave upward. Growth in the filamentous fungi is limited to the tips of the hyphae. The influence of neighboring cells which compete for nutrients is a much more important factor in the growth of filamentous fungi than in submerged unicellular organisms. In unagitaged cultures a portion of the mycelium is usually aerial at some stage of growth. The aerial mycelium derives its nutrients from the submerged cells, which involves the transport of these substances over some distance. RATE OF GROWTH To study growth, it is necessary to consider both the rate and amount of production of cells formed during incubation. The average rate of growth is obtained by measuring the amount of growth at two intervals of incubation and dividing the difference by the time interval. If the weight of a fungus colony increased from 50 to 98 mg. between the fourth and sixth days of incubation, the average rate of growth is 24 mg. per day, or 1 mg. per hr. In experimental work, measurements of growth should be made sufficiently often during the period of incubation so that a smooth graph (growth curve) can be plotted from the data. The inter- vals between measurements of growth may be as short as 1 day for a rapidly growing fungus and as long as a week for species which grow slowly. The rate of growth at any time may be determined by finding the slope (tangent) of the curve. The growth rates of fungi differ, as is illustrated in Fig. 2. Since growth is a process which takes place in time, it can be studied only by making many growth measurements during the period of incuba- tion. Such a study is not complete until the phase of autolysis is attained. Much of the information in the literature is incomplete because growth was measured only at one time. Many of the potentialities of the fungi can be discovered only by prolonged observation. GROWTH 27 WAYS OF MEASURING GROWTH The discussion of phases of growth presupposes methods of measuring growth. In choosing a method of measuring growth, or any other physio- logical process, the accuracy and type of information desired must be kept in mind. For some purposes the simplest methods are satisfactory; for others the most accurate methods should be chosen. Visual inspection. The simplest way to measure growth is by inspec- tion and comparison. The value of this method lies in the speed with which growth measurements are made. Elaborate equipment is not 400 300 • 200 T5 'o S too 16 18 20 4 6 8 10 12 14 Doys of incubation Fig. 2. Growth of four fungi under the same conditions, in 25 ml. of liquid glucose- casein hydrolysate medium at 25°C. needed, as test tubes and Petri dishes are satisfactory culture vessels. This method has the further advantage that the same cultures may be kept under observation. It is frequently the method of choice for pre- liminary experiments, for the very appearance of the mycelium is a clue to the amount of growth. Growth under varying conditions may be compared if some condition is used as a standard for comparison (see Suggested Laboratory Exercises). It is obvious that a great deal of sub- jective judgment enters into this method of estimating growth, but it is veiy useful where fine distinctions are not required. Linear growth. A second widely used method of measuring growth consists in growing fungi in Petri dishes and measuring either the diameter or the area of the colony. This is a useful method in some instances but 28 PHYSIOLOGY OF THE FUNGI almost useless in others. At least these measurements can be made in an objective way. In this method, the diameter, radius, or area of a colony is used to express the amount of growth, while the daily increase repre- sents the rate of growth. It is obvious that this method neglects the thickness of the colony. Worley (1939) has proposed to take the thick- ness of the mycelium into account when growth is measured by this method. Such measurements are difficult and neglect the mycelium buried in the agar. The rate of linear growth of some fungi has little relation to the composition of the medium. The rapid extension of mycelium on water-agar medium may serve as a familiar example. It has been frequently assumed that fungi grow at a constant rate when maintained under constant environmental conditions. This assumption is not necessarily true, for the growth of Aspergillus rugulosiis and many other fungi is self-limited under cultural conditions. Two factors may contribute to cause nonuniform rates of growth: (1) the change in con- centration of nutrients due to diffusion and utilization; (2) the excretion of inhibitory metabolic products into the medium. The same fungus may have a constant rate of growth at one tempera- ture and not at another. The rate of growth is frequently not constant when fungi are cultured at temperatures higher than optimum. Fawcett (1921) found the rate of growth of Phytiacystis citrophthora, Phytophthora terrestris, Phoviopsis citri, and Diplodia natalensis to decrease with time when these fungi were cultivated above the optimum temperature. Some of Fawcett's data which illustrate this phenomenon are given in Table 4. Table 4. The Effect of Temperature upon the Rate of Growth OF Three Fungi The daily increase in the average radius of the colonies is given in milhmeters, (From the data of Fawcett, Univ. Calif. {Berkeley) Pubs. Agr. Sci. 4, 1921.) Phytiacystis Phytophthora Phomopsis Days of incubation citrophthora terrestris citri 23.5°C. 31.0°C. 30.0°C. 35.5°C. 27.5°C. 32.0°C. 1 5.4 6.3 5.5 4.8 4.6 0.9 2 10.0 5.5 13.8 4.2 8.0 0.3 3 10.2 3.5 13.3 2.6 8.0 0.2 X 10.5 1.5 13.2 2.5 8.5 0 5 10.5 0.5 10.9 0 8.5 0 If the rate of growth under a given condition does not change with time, this method is useful and simple. It permits observation of the same culture for the duration of the experiment. Ryan et al. (1943) have proposed the use of an ingenious growth tube in which linear growth can GROWTH 29 be measured with ease and accuracy. This growth tube is illustrated in Fig. 3. These authors (Ryan et al, 1943) found the rate of linear growth of Ncurospora sitophila in such a growth tube to be constant for 200 hr. The growth-tube method has been used to study the effect of temperature, pH, vitamin content, and other variables upon Neurospora. These special tubes have another advantage over Petri dishes in that cultures are well protected from contamination. The same culture may be exposed to a variety of environmental conditions such as hght and tem- perature. These tubes have the disadvantage that it is more difficult to remove mycelium or fruit bodies for examination. In addition, aeration may be poor and become a limiting factor for some fungi. Fig. 3. Growth tube patterned after those described by Ryan, Beadle, and Tatum {Am. Jour. Botany 30: 784-799, 1943) for measuring linear growth. Dry weight. By weighing the mycelium and spores produced, an accurate and objective measure of growth is obtained. For precise work it is the method of choice. Where any significant weight of spores is pro- duced, either Gooch or Alundum crucibles may be used to collect both mycelium and spores. For most purposes the mycelium may be filtered from the culture medium by use of a finely woven cloth and then trans- ferred to weighing bottles or small aluminum cups. The excess medium should be removed by washing and pressing the mycelium, which is then dried to constant weight at 80 to 100°C. After the mycelium is dry, it is weighed on an analytical balance. It is usually sufficient to record the weight to the nearest milligram. Some fungi make better growth and sporulate more readily on agar than in liquid medium. It is desirable to have an objective measure of growth of agar cultures. Fries (1943) and Day and Hervey (1946) have obtained the dry weight of cultures grown on agar. This technique should be more widely used. The mycelium is freed from agar by briefly autoclaving the cultures, filtering off the mycelial mats, and washing with :36 PHYSIOLOGY OF THE FUNGI hot water. Frequently the mat can be removed from the melted agar with a pair of forceps instead of by filtering. Autoclaving removes some soluble constituents from the mycelium, but if a uniform procedure is adopted, the results are comparable. Measuring yeast growth. The growth of yeasts may be measured by four methods. (1) Yeast cells may be counted in an aliquot of the medium by the use of a hemocytometer or other counting chamber. The method is tedious. (2) The volume of yeast cells in a given volume of medium may be measured in special graduated centrifuge tubes. Yeast 80r 3.1 6.2 0.8 1 .6 iig. fhiomine per culture Fig. 4. Direct comparison between diameters and dry weights of the same 10-day- old cultures of Ceratostomella fimbriata in the presence of varying amounts of thiamine. Cultures were grown in Petri dishes on 25 ml. of glucose-casein hydro lysate agar at 25°C. cells are large and easily separated from the medium by centrifuging. This method is less tedious than counting. (3) Turbidity may be used to measure the amount of yeast growth. Accurate determinations by this method require the use of a photoelectric photometer. This method is rapid and sufficiently accurate for many purposes. Lindegren and Raut (1947) have cultivated yeasts in colorimeter tubes and have followed the rate and amount of growth for as long as desired. (4) Yeast cells may be filtered under vacuum, washed, dried, and weighed. Selas porcelain crucibles with fritted bottoms are suitable. This method is accurate but somewhat time-consuming. Comparison of methods. It should be clearly recognized that one method of measuring growth may not agree with another. This is illus- GROWTH 31 trated by Fig. 4, where two methods of measuring the amount of growth of Ceratostomella fimbriata were used. This figure demonstrates that the diameter of a colony may be a very poor measure of the amount of growth. Fries (1943) grew Ophiostoma {Ceratostomella) ulmi on agar medium and measured the radii of the colonies and also w^eighed the mycelium after removing the agar. After 5 days the average radius of cultures without pyridoxine was 16.3 mm., while the average radius of cultures receiving pyridoxine was 12.3 mm; the weights of mycelium produced under these two conditions were 5.2 and 18.1 mg., respectively. It is clear from these examples that different methods of measuring growth do not always give comparable results. Before valid conclusions can be reached, it is neces- sary to use valid methods of measuring the quantities involved. METHODS OF PRESENTING RESULTS The data obtained in a well-planned and carefully executed experiment have value in themselves, but more frequently data are a means to an end. Experimental data form the basis upon which conclusions are reached and serve as a guide to further investigation. A conclusion is sound only if the data are sound. To be of greatest value, data must be presented in an understandable manner. Extensive data may be presented either as tables or graphs; each method has certain advantages. Tables. The utility and conciseness of tables make them desirable for many purposes. Tables are especially suitable in comparing the amount of growth (or any other function under study) of a number of fungi under standard conditions or under a number of conditions. They give the reader the same basic and fundamental information available to the original investigator. The utility of such information can be appreciated only when one attempts to assess the reports in the literature. Derived data, such as ratios or percentages, may be needed for the pur- poses of interpretation and study, and as such they are entirely proper. However, the original data from which the derived data were calculated should always be published. The original data frequently have values which are not perceived or considered by the original investigator. Derived data as such afford no clue as to the original magnitudes. Without the original data no comparison can be made with other experi- ments, whether in the same or other laboratories. The usefulness of many publications is severely limited because the author presented only ratios or percentages instead of the original data. If a datum represents an average value, the number of determinations upon which it is based should be stated. It is desirable to indicate the range of variation among replicates, or the standard deviation should be given if the number of observations is large. 32 PHYSIOLOGY OF THE FUNGI Graphs. The significance of data is frequently best appreciated when presented in graphical form. A graph reminds one that growth is a con- tinuous function in time, whereas a table may suggest a discontinuous process. Growth curves are especially suited to illustrate the rate and amount of growth as a function of time. In Fig. 2 the growth curves of four fungi illustrate differences among species. Growth curves are equally applicable to the study of a single species under different condi- tions. The points representing the data should be given, so that the reader may see how closely the curve fits the data. Three-dimensional graphs may be used to represent the relations among three variables. Three-dimensional graphs take the form of a surface. Rahn (1939) has given concise directions for constructing such graphs and models. Schopfer (1943) has used such graphs to represent the growth of Phycomyces hlakesleeanus with respect to the amount of thiamine and asparagine in the medium as a function of time of incubation (Fig. 33). Another way of showing the relations among the variables involves the use of a triangular graph. Such a presentation is effective if one desires, for example, to show the effect of the concentrations of three constituents of a medium upon growth. For examples of the use of triangular graphs see Haenseler (1921) and Pratt and Hok (1946). Photographs. The presentation of experimental results is frequently improved by the judicious use of photographs. Photographs are particularly useful in comparing the behavior of fungi under different experimental conditions. The behavior of different species under identical conditions may be effectively compared by the use of photo- graphs. Well-labeled photographs also make excellent permanent records of certain types of experimental results. FACTORS AFFECTING GROWTH All the separate factors comprising the internal and external environ- ment may affect either the rate or the amount of growth, or both. Among the internal factors are the genetic constitution and the internal modifica- tions due to age and to the previous external environment. While more is known about the external factors which affect growth than about the internal factors, it should always be remembered that the external envi- ronment acts by modifying the internal environment. Internal factors. One species differs from another, and even one isolate of a species may differ from another in genetic composition. Many mutations have been produced in the laboratory by the action of X rays, ultraviolet rays, and certain chemicals (see Chaps. 10 and 18). These mutants of a single species produced in the laboratory differ from the parent type in one or more biochemical or morphological characteristics and thus correspond to the different isolates of a species found in nature. GROWTH 33 There is no reason to suppose that mutants produced in the laboratory- differ fundamentally from those isolated in nature. The potentiahties of a fungus are limited by its genetic constitution. The realization of these potentialities may be denied or favored by the external environment, and only as the environment is suitable do these inherent factors find expression. Diversity, rather than uniformity, in behavior among species and isolates is the rule. Only a small amount of inoculum is used in most studies. It is impor- tant to learn if the age, history, or kind of inoculum has any effect on the subsequent development of the fungus. All these factors may influence the rate and amount of growth and other functions of the fungi. Young and vigorously growing inoculum is most suitable, since old cells as a general rule are slow to start growth. Apparently one of the first func- tions a cell loses is the power of division. From this standpoint such cells are "dead," although they may be still capable of performing many vital functions, such as respiration. Difficulty is frequently experienced in making subcultures from old cultures. Certain species are difficult to maintain in culture unless they are frequently subcultured. In general, these species do not readily form resting cells. Among these are various species of Pythium and Phytophthora, test-tube cultures of Choanephora cucurhitarum, and others. In experimental work of the highest precision neither the temperature nor the medium upon which the inoculum is grown may be neglected. Zikes (1919) investigated the generation time of six strains of yeast and found that the storage temperature of the inoculum affected the time required for cell division. These original cultures were grown at 8°C. and 25°C., and subcultures were incubated over a range of temperatures. When the inoculum which was grown and stored at 8°C. was subcultured at low temperatures, the generation time was less than that of the culture grown and stored at 25°C. At temperatures above 25°C. the generation time of the high-temperature yeast was less than that of the low-tempera- ture yeast. In some way, yeast cells cultured over long periods of time at a certain temperature become adapted to this temperature, and when such cells are transferred to other temperatures, the influence of the original temperature of incubation persists for a time. It is evident that some change in the internal environment has occurred. Comparable studies on the filamentous fungi are rare. From Fawcett's data on the rate of linear growth of four citrus pathogens it appears that the same phenomenon takes place with some filamentous fungi. Fawcett grew the inoculum at 20°C., and on subculturing at 7.5°C. the linear rate of growth increased with time, as is shown in Table 5. Many fungi have latent abilities to synthesize various essential metab- olites. In the virtual absence of these compounds in the medium and 34 PHYSIOLOGY OF THE FUNGI after a shorter or longer period of incubation, a fungus may begin to synthesize these essential metabolites, and growth then takes place in a normal way. This is especially true of the yeasts with respect to vitamins. Many fungi lose their pathogenicity Avhen cultured for a long time on laboratory medium. Host passage frequently restores pathogenicity. The indiscriminate use of inoculum from a variety of substrates and of different ages may introduce unexpected variation in experimental work and should be guarded against. Table 5. Daily Increase (in Millimeters) in Diameter of Colonies OF Four Fungi Inoculum grown at 20°C.; subcultures incubated at 7.5°C. (From the data of Fawcett, Univ. Calij. {Berkeley) Pubs. Agr. Sci. i, 1921.) Species Phythiacystis citrophthora Phytophthora terrestris . . . Phomopsis citri Diplodia natalensis 1st day 0.04 0.02 0.01 0.05 2d day 0.4 0.14 0.16 1.9 3d day 0.6 0.21 0.83 2.1 4th day 0.8 0.7 0.9 5th day 1.2 0.8 1.0 External factors. Among the external factors which influence the growth of fungi, temperature plays an extremely important role. Tem- perature affects almost every function of the fungi. For each fungus there is a temperature below which it will not grow, the minimum tem- perature. Likewise there is a temperature above which growth ceases, the maximum temperature. These two temperatures indicate the tem- perature range of an organism. A few fungi are capable of growing below 0°C., but for most species the minimum temperature is 0 to 5°C. The maximum temperature varies from 27°C. ior Phacidium infestans (Pehrson, 1948) and Sclerotinia cameUiae (Barnett and Lilly, 1948) to 45 or 50°C. for Aspergillus fumigatus (Thom and Raper, 1945). The maximum tempera- Table 6. Cardinal Temperatures for Various Fungi Species Minimum, Optimum, °C. Maximum, °C. Citation Neurospora sitophila Ceratostomella pilifera C. ips 4 5 5 -3 2 12.0 0.5 36 25-30 30 15 18-21 31.5 25-30 44 35 40 27 26 36.1 40 Ryan et al, 1943 Lindgren, 1942 Lindgren, 1942 Phacidium infestans Phytophthora infestans P. terrestris Pehrson, 1948 Crosier, 1933 Fawcett, 1921 Various yeasts Zikes, 1919 GROWTH 35 ture is sometimes an important factor limiting the attack of plant pathogens. The cardinal temperatures of a few fungi are given in Table 6. A more extensive compilation is given by Wolf and Wolf (1947). The character- istic effect of different temperatures on the rate of growth of two fungi is shown in Fig. 5. Further examples may be found in the work of Lindgren (1942). Most reports on the effect of light on the fungi have been concerned with reproduction rather than vegetative growth. However, Elfving (1890) found strong diffuse daylight to depress the growth of Penicillium glaucum and a species of Briarea. The amount of inhibition was least when the culture medium contained complex nutrients such as peptone. Greater inhibition resulted when the media contained glucose, mannitol, and malic acid. Scattered observations indicate that the depressing effect of strong light may be rather common. In the old literature some mention is made of the favorable effect of light on red yeasts. The sporangiophores of Phycomyces hlakesleeanus attain a greater length in darkness than in intense light. The role of light in the sporulation of some fungi is discussed in Chap. 14. Conclusive evidence that light affects the amount of growth of Karlingia (Rhizophijlctis) rosea, one of the lower Chytridiales, was presented by Haskins and Weston (1950). This fungus when grown in liquid glucose- nitrate medium produced twice the amount of dry weight of cells when cultured in light than when the cultures were kept in total darkness. With the exception of the factor of illumination, the experimental con- ditions were the same. Approximately twice as much glucose was utilized by cultures exposed to light as those kept in darkness. On the other hand, when K. rosea was grown in a liquid cellobiose-nitrate medium, more growth resulted in total darkness than in light. The explanation for this behavior of K. rosea is not known. The moisture requirements of fungi differ. Most species in nature live on substrates which are not saturated with water. The low moisture content of a substrate is often a factor which limits the growth of fungi. Particularly is this true of the species which live on wood or in soil. As a general rule, wood which contains less than 20 per cent moisture is immune to fungus decay. A difference of a few per cent in the moisture content may determine whether a species will be able to grow or not. Lindgren (1942) has reported that Ceratostomella pilifera, a wood-staining fungus, does not grow in pine wood having a moisture content of 23 per cent but develops in wood containing 24.5 per cent moisture. The maximum rate of penetration was attained on wood having a moisture content of 29 per cent or more. Jute sacking is subject to fungus attack only if the mois- ture content exceeds 17 per cent. 36 PHYSIOLOGY OF THE FUNGI In physiological studies dealing with high concentrations of nutrients, it is important to distinguish between osmosis and osmotic pressure. Osmosis is the transfer of water through a membrane permeable to water 160 120 ^80 E 40 ^ i"^"^ ^^^ \ / \ \ \ 1 \ \ / / - 10 20 Degrees centigrade 30 40 10 20 30 Temperature in degrees centigrade Fig. 5. A, the effect of temperature on the dry weight of mycelium produced by Glomerella cingulata after 5 days in 25 ml. of liquid glucose-asparagine medium. (Drawn from the data of I. G. Bennett, 1951.) B, the effect of temperature on the rate of linear growth of Neurospora crassa. (Courtesy of Ryan, Beadle, and Tatum, Am. Jour. Botany 30 : 785, 1943.) but not to the solute molecules. In simple systems water passes from a dilute to a more concentrated solution. Osmotic pressure is the force necessary to restrain the movement of water from a dilute to a concen- trated solution through a semipermeable membrane. The osmotic pres- sure which a solution is capable of developing is a function of the number GROWTH 37 of ions and molecules of solute contained in a unit \'olume of solution. A mole of a non-ionized compound in 1,000 g. of water at 0°C. has an osmotic pressure of 22.4 atm. if separated from pure water by a semiper- meable membrane. For a fuller discussion of osmosis and osmotic pres- sure the student is referred to Gortner (1949), Seifriz (1936), and Meyer and Anderson (1948). If concentration were the sole factor which determines whether growth is possible, all solutions having the same osmotic pressure would be equally inhibitory. Table 7 indicates that this is not true. Table 7. Highest Osmotic Pressures (Atmospheres) op Solutions of Four Compounds in Which Various Fungi Grew (Hawkins, Jour. Agr. Research 7, 1916.) Species Glucose* Sucrose Potassium nitrate Calcium nitrate Plenodomus destruens Diplodia tubericola Rhizopus nigricans Botrytis cinerea .... 58.3 63.2 63.2 63.2 63.2 47.4 42.1 42.1 47.4 47.4 54.5 58.8 27.5 54.5 54.5 33.6 33.6 15.9 27.7 Ceratoslomella fimhriata 19.5 * Limiting concentrations not used. These data and others show that the limiting osmotic pressure depends upon the fungus and the compounds used. It is difficult to evaluate the effects of osmotic pressure upon the fungi, for the cell membrane is per- meable to other compounds in addition to water. Calculations of osmotic pressure are made by assuming that an indifferent semipermeable mem- brane separates solutions of different concentrations. The effect of osmotic pressure upon the fungi cannot be considered as a simple physio- chemical process. However, the ability of many fungi to grow in solu- tions having high osmotic pressures is advantageous. Parasitic fungi characteristically have a higher osmotic pressure than the cell sap of the plants they parasitize (Thatcher, 1939). For further references to the effect of osmotic pressure on fungi, see Kroemer and Krumbholz (1931). Another process involved in the entrance of water into fungus cells is imbibition. Gortner (1949) has defined imbibition as the process whereby colloidal substances such as protoplasm take up water, and imbibition pressure as the pressure against which a colloid will imbibe liquid. Raciborski (1905) grew a species of Torula in saturated lithium chloride (1,000 atm.) and Aspergillus glaucus in a saturated sodium chloride solution. Aside from osmotic effects, the concentration of the medium has a great effect on the rate and amount of growth of fungi. The concentration of 38 PHYSIOLOGY OF THE FUNGI nutrients which is most favorable for growth may be poor in other respects, e.g., for reproduction. The concentration may be varied in two ways: (1) by dihiting the entire medium, whereby the ratios among the constituents remain unchanged, and (2) by varying the concentration of one constituent. These methods are not equivalent and yield different results. When an entire medium is diluted, it might be expected that the de- crease in amount of mycelium produced would be directly proportional to the amount of dilution. Such is not always the case. When Chae- tomium convolutum was grown in full-strength medium and in medium diluted to one-fourth and one-sixteenth full strength, the maximum weights of mycelium produced were 220, 75, and 22 mg., respectively (Lilly and Barnett, 1949). C convolutum grew most efficiently in the most dilute medium. This principle appears to be generally valid and is also illustrated by Ceratostomella fimbriata (Table 57) . Table 8. The Effect of Different Volumes of Medium upon the Rate and Maximum Amount of Growth of Sordaria fimicola Dry weight of mycelium in milligrams. Days of Ml. medium per 250-ml. Erlenmeyer flask incubation 6.25 12.5 25.0 50.0 3 47 80 63 22 4 75 99 129 99 5 71 113 166 160 6 65 100 156 238 9 57 107 168 269 When the concentration of one constituent in the medium is changed, over a certain range, the amount of growth will be proportional to the concentration. Above a certain concentration there will be no further increase in the amount of growth. This is due to the limiting concentra- tion of some other constituent in the medium. This is the principle upon which fungi are used in vitamin and other assays (Chap. 10). The maximum weight of mycelium which is obtained from a given vol- ume of medium depends upon the type and size of the culture vessels used. The rate of growth is also affected. These results appear to be due mainly to differences in aeration, and perhaps to a lesser degree to diffu- sion. The effect of depth of medium on rate and amount of growth in non- agitated cultures may be demonstrated by using a constant volume of medium in different-sized flasks, or by varying the volume of medium in flasks of the same size. Data illustrating this latter condition are pre- sented in Table 8. The slow initial rate of growth when the mycelium is GROWTH 39 entirely submerged is due to lack of an adequate supply of oxygen. The efficiency of Sordaria fimicola in converting the constituents of the medium into mycelium decreased as the depth of the medium increased. This fungus was less than half as efficient when grown in 50 ml. of medium as when grown in 6.25 ml. EFFECT OF EXTERNAL FACTORS ON MORPHOLOGY While the study of morphology, as such, is not within the province of physiology, there is a close connection between these two aspects of mycology. Form and function are the two ways in which the poten- tialities of organisms come to expression. The morphology of a fungus may be modified by environmental factors to such a degree as to be unrecognizable. These changes in morphology may be microscopic as well as grossly visible. Pasteur (1879) noted that species of Mucor, when grown submerged in liquid and in the absence of air, assumed a yeast-like form. Not only did they resemble yeasts, but under these conditions they fermented sugar to alcohol. Under aerobic conditions no detectable amounts of alcohol were formed. Reproductions of Pasteur's drawings have been published by Foster (1949). When yeasts are cultured in liquid media and allowed to age undis- turbed, a film or membrane frequently covers the surface of the liquid. Film formation frequently starts as a ring of cells on the wall of the flask at the air-liquid interface. The morphology of the yeast cells in such films is unusual in that the cells are joined together in filaments. The supply of oxygen must play an important role in the formation of fila- ments. The temperature range within which film formation occurs varies with the species of yeast and is usually considerably less than the tempera- ture range for growth. Most species of yeasts forms films only between 6 and 30°C., although Zikes (1919) found Monilia Candida and Mycoderma cerevisiae to form films at 37°C. The early literature on this subject has been summarized by La Far (1911). Nickerson and Van Rij (1949) have reviewed the mechanisms of fila- ment formation in yeast and conclude that the processes of cell elongation and cell division are controlled by different enzyme systems. Appar- ently, the sulfhydryl enzymes which regulate the process of cell division may be inhibited without greatly interfering with cell elongation. Among the agents which inhibit cell division are cobalt, iodoacetate, and peni- cillin. The effect of penicillin on Saccharomyces cerevisiae is shown in Fig. 6. Camphor and other narcotizing agents produce somewhat the same changes in morphology of yeast cells (Levan, 1947). Many pathogenic fungi which cause disease in man are dimorphic. These fungi are usually yeast-like in the host but frequently form myce- 40 PHYSIOLOGY OF THE FUNGI Fig. 6. Saccharomyces cerevisine, camera lucida drawings of cells from agar cultures. A, culture treated with penicillin; B, culture treated with penicillin plus cysteine. (Courtesy of Nickerson and Van Rij, Biochim. et Biophijs. Acta 3: 461-475, 1949. Published by permission of Elsevier Book Company, Inc.) iw \ . \*' i/^/\ "" A B Fig. 7. The effect of hydrogen-ion concentration on the morphology of cells of Sordaria fimicola. A, rounded swollen cells produced in glucose-casein hydrolysate medium at initial pH 3.6. B, normal mycelium from the same culture a few days after a drop of NaOH was added. Hum in culture. Blastomyces dermatitidis and B. hrasiliensis exhibit thermal dimorphi.sm (Nickerson and Edwards, 1949). When these fungi are cultured on certain media at 37°C., they are yeast-like, while at lower temperatures of incubation they form mycelium. This change in mor- phology is accompanied by changes in the rate of respiration and type of GROWTH 41 Fig. 8. The effect of environment on the morphology of fruit bodies of Forties applanatus. A, normal fruit body developed in nature; B, C, malformed fruit bodies of the same (?) fungus developed under water in abandoned coal mines. The "nodes " in B are believed to be caused by different water levels. 42 PHYSIOLOGY OF THE FUNGI metabolism. Chemical agents may favor or prevent similar morphologi- cal changes. Trichophyton ruhrum produces two metabolic products of unknown constitution which inhibit the transformation of Candida albicans to the mycelial form (Jillson and Nickerson, 1948). The addi- tion of excessive amounts of inositol to the culture medium causes Ophiostoma (Ceratostomella) muUiannulatum to grow almost entirely in the form of conidia (Fries, 1949). The morphology of the vegetative mycelium and sporangia of various species of Phytophthora was found to depend upon the medium used (Leonian, 1925). The form of mycelial growth of many species, when grown on agar media, is an aid in identification. The colony form may be altered beyond recognition when cultures are grown in agitated liquid medium. In general, spherical colonies or balls form in agitated medium. Burk- holder and Sinnott (1945) investigated colony form of a large number of species when subjected to agitation. The acidity of the medium affects the size and shape of the vegetative cells of some fungi. In a medium so acid as to allow only very slow growth the cells often become swollen or nearly spherical in shape, much like chlamydospores, but the wall remains thin (Fig. 7). This may be accompanied by excessive branching. Unusual environmental conditions often affect the morphology of both vegetative and reproductive structures. The environment which exists in coal mines is unnaturally uniform with respect to temperature, mois- ture,'and absence of light. Basidiomycetes growing on old mine timbers either fail to fruit or produce odd-shaped sterile fruit bodies (Fig. 8). SUMMARY Normal growth results in an increase in cell number and mass. Limited growth may result from either of these two processes alone. Growth is a phenomenon which requires time for its various manifestations. Growth follows a pattern which differs from species to species, but the general sequence of phases is much the same for all fungi. Growth studies are based upon measuring both the amount and the rate of growth. The rate and amount of growth are controlled by the internal and external environ- ment. The potentialities of a fungus are limited by its genetic constitu- tion, but the expression of these potentialities is controlled by external factors such as temperature, light, composition, and concentration of the medium. Even the size and shape of the culture vessels used affect the rate and amount of growth. The amount of growth can be estimated by visual comparison or meas- ured by determining the diameter of a colony or by harvesting the myce- lium and weighing it after drying to constant weight. The amount of yeast growth may be measured by counting the numbers of cells produced, GROWTH 43 by centrifuging and measuring the volume of cells, by turbidity, or by weighing. The most direct way of measuring growth of either yeast or filamentous fungi is by weighing the crop produced. The various meth- ods of measuring growth are not strictly comparable. The morphology of a fungus may be changed by environmental factors so that it becomes unrecognizable. The processes of cell elongation and cell division are controlled by different enzyme systems. In some instances it has been possible to inhibit cell division without interrupting cell elongation. Frequently a change in physiology accompanies a change in morphology. REFERENCES *Barnett, H. L., and V. G. Lilly: The interrelated effects of vitamins, temperature, and pH upon vegetative growth of Sclerotinia camelliae, At7i. Jour. Botany 35: 297-302, 1948. Bennett, I. G.: Thesis, West Virginia University, 1951. Buchanan, R. E., and E. I. Fulmer: Physiology and Biochemistry of Bacteria, Vol. I, The Williams & Wilkins Company, Baltimore, 1928. Burkholder, p. R., and E. W. Sinnott: Morphogenesis of fungus colonies in sub- merged shaken culture. Am. Jour. Botany 32: 424-431, 1945. Crosier, W.: Studies in the biology of Phytophthora infestans (Mont.) de Bary, Cornell Univ. Agr. Expt. Sta. Mem. 155, 1933. Day, D., and A. Hervey: Phycomyces in the assay of thiamine in agar. Plant Physiol. 21 : 233-236, 1946. Elfving, F.: Studien iiber die Einwirkung des Lichtes auf die Pilze, Helsingfors Central-Druckerei, Helsingfors, 1890. Emerson, S.: The growth phase in Neurospora corresponding to the logarithmic phase in unicellular organisms, Jour. Bad. 60: 221-223, 1950. *Fawcett, H. S. : The temperature relations of growth in certain parasitic fungi, Univ. Calif. (Berkeley) Pubs. Agr. Sci. 4: 183-232, 1921. Foster, J. W.: Chemical Activities of Fungi, Academic Press, Inc., New York, 1949. Fries, N.: Die Einwirkung von Adermin, Aneurin und Biotin auf das Wachstum einiger Ascomyceten. Symbolae Botan. Upsalienses 7(2): 1-73, 1943. Fries, N.: Ophiostoma multiannulatum (Hedge, and Davids) as a test object for the determination of pyridoxin and various nucleotide constituents. Arkiv fiir Botanik 1: 271-287, 1949. Gortner, R. a.: Outlines of Biochemistry, 3d ed., John Wiley & Sons, Inc., New York, 1949. Haenseler, C. M.: The effect of salt proportions and concentrations on the growth of Aspergillus niger, Am. Jour. Botany 8: 147-163, 1921. *Haskins, R. H., and W. H. Weston, Jr.: Studies in the lower Chytridiales. I. Fac- tors affecting pigmentation, growth, and metabolism of a strain of Karlingia (Rhizophlyctis) rosea, Am. Jour. Botany 37 : 739-750, 1950. Hawkins, L. A.: Growth of parasitic fungi in concentrated solutions, Jour. Agr. Research!: 255-260, 1916. JiLLSON, O. F., and W. J. Nickerson: Mutual antagonism between pathogenic fungi. Inhibition of dimorphism in Candida albicans, Mycologia 40: 369-385, 1948. Kroemer, K., and G. Krumbholz: Untersuchung iiber Osmophile Sprosspilze. I. Mitteilung, Beitrage zur Kenntniss der Garungsvorgange und der Garungs- erreger der Trockenbeerenauslesen, Arch. Mikrobiol. 2: 352-410, 1931. 44 PHYSIOLOGY OF THE FUNGI La Far, F.: Tochnical Mycology. Vol. II, Eumycetic Fermentation (trans. C. T. C. Salter), Chas. Griffin & Co., Ltd., London, 1911. Leonian, L. H.: Physiological studies on the genus Phytoyhthora, Am. Jour. Botany 12:444-498, 1925. Lev AN, A.: Studies on the camphor reaction of yeast, Heredilas 33: 457-514, 1947. Lilly, V. G., and H. L. Barnett: The influence of concentrations of nutrients, thiamin, and biotin upon growth and formation of perithecia and ascospores by Chaetomium convolutum, Mycologia 41 : 186-196, 1949. LiNDEGREN, C. C, and C. Raut: The effect of the medium on apparent vitamin- synthesizing deficiencies of microorganisms. A direct relationship between pantothenate concentration and the time required to induce the production of pantothenate-synthesizing "mutants" in yeasts, Ann. Missouri Botan. Garden 34 : 75-90, 1947. LiNDGREN, R. M. : Temperature, moisture, and penetration studies of wood-staining Ceratostomellae in relation to their control, U.S. Dept. Agr. Tech. Bull. 807, 1942. Meyer, B. S., and D. B. Anderson: Plant Physiology, D. Van Nostrand Company, Inc., New York, 1948. NiCKERSON, W. J., and G. A. Edwards: Studies on the physiological bases of morphogenesis. I. The respiratory metabolism of dimorphic pathogenic fungi, Jour. Gen. Physiol. 33: 41-55, 1949. NiCKERSON, W. J., and N. J. W. van Rij: The effect of sulfhydryl compounds, penicillin, and cobalt on the cell division mechanism of yeasts, Biochim. et Biophys. Acta 3: 461-475, 1949. *Pasteur, L.: Studies on Fermentation. The Diseases of Beer, Their Causes, and the Means of Preventing Them (trans, from Etudes sur la biere by F. Faulkner and D. C. Robb.), Macmillan & Co., Ltd., London, 1879. Pehrson, S. O.: Studies of the growth physiology of Phacidium infestans Karst., Physiologia Plantarum. 1 : 38-56, 1948. Pratt, R., and K. A. Hok: Influence of the proportions of KH2PO4, MgS04, and NaNOs in the nutrient solution on the production of penicillin in submerged cultures, Am. Jour. Botany 33: 149-156, 1946. Raciborski, M.: Ueber die obere Grenze des osmotischen Druckes der lebenden Zelle, Bull, internat. acad. sci. Cracovie, CI. sci. math, et nat. 7: 461-471, 1905. Rahn, 0.: Physiology of Bacteria, The Blakiston Company, Philadelphia, 1932. Rahn, O.: Mathematics in Bacteriology, Burgess Publishing Co., Minneapolis, 1939. Ryan, F. J., G. W. Beadle, and E. L. Tatum: The tube method of measuring the growth rate of Neurospora, Atn. Jour. Botany 30: 784r-799, 1943. ScHOPFER, W. H.: Plants and Vitamins, Chronica Botanica Co., Waltham, 1943. Seifriz, W.: Protoplasm, McGraw-Hill Book Company, Inc., New York, 1936. *Thatcher, F. S.: Osmotic and permeability relations in the nutrition of fungus parasites. Am. Jour. Botany 26: 449-458, 1939. Thom, C, and K. B. Raper: A Manual of the Aspergilli, The Williams & Wilkins Company, Baltimore, 1945. Wolf, F. A., and F. T. Wolf: The Fungi, Vol. II, John Wiley & Sons, Inc., New York, 1947. WoRLEY, C. L.: Interpretation of comparative growths of fungal colonies on different solid substrata. Plant Physiol. 14 : 589-593, 1939. ZiKEs, H.: Ueber den Einfluss der Temperature auf verschiedene Funktionen der Hefe, Cent. Bakt., Abt. II, 49: 353-373, 1919. CHAPTER 4 ENZYMES AND ENZYME ACTION The fungi, in common with other hving organisms, possess tools or reagents far more specific, more deHcate, and more powerful than those available in the laboratory. The most complex natural substances such as proteins, polysaccharides, and Hpoids are degraded into simpler com- pounds which are soluble in water. Fungi also synthesize similar com- plex compounds from relatively simple molecules. These transformations are carried out under such mild conditions of temperature and pressure and in such low concentrations of acid and alkali that it is certain the means used are of a peculiar kind. For in the absence of these special agents formed by the living organisms, these reactions do not take place or do so at a very slow rate. These organic catalysts produced by living organisms are called enzymes. The life processes of organisms are con- trolled and directed by a complicated and interrelated series of enzymes or enzyme systems (Dixon, 1949). Some enzymes formed by fungi are excreted and normally perform their functions outside the cells that produce them. These are termed exo- enzymes (extracellular enzymes), such as cellulase, amylase, and pectinase. Exoenzymes perform the functions of digestion; i.e., the degradation of complex food materials into low-molecular-weight compounds which are able to enter the cell. After entering the cell, these metabolites are acted upon by the enzymes within the cell. These enzymes are called endo- enzymes (intracellular enzymes). Naturally enough, exoenzymes were recognized and studied first. In the early literature, these exoenzymes were called unorganized ferments because of their solubility. In contrast to these unorganized ferments it was recognized that other ferments (enzymes) occurred in an insoluble organized form. These were called organized ferments. Pasteur (1875) still spoke of yeast as "ferment alcoohque ordinaire du vin." Thus, the name organized ferment took on a dual meaning, that of a living organism and the various chemical reactions caused by these organisms. In 1878 Kiihne suggested that the word enzyme be used to replace the terms organized and unorganized ferments. Enzyme is derived from the Greek phrase, en zyme, which means in yeast or leaven. For excellent sum- maries of the historical development of the relation between fermentation 45 46 PHYSIOLOGY OF THE FUNGI and the action of microorganisms, see Stephenson (1939) and Harden (1932). It was not until late in the nineteenth century that Buchner (1897) succeeded in releasing certain enzymes from yeast cells and demonstrating that the endoenzyme(s) in yeast causing fermentation was also active entirely apart from the living yeast cells. While yeast juice prepared according to the method of Buchner contained a variety of enzymes, it contained fermentative enzymes never before obtained apart from the living cell. These enzymes cleaved sugar into alcohol and carbon dioxide. This was truly a monumental step in the science of enzymes, for it afforded a way of studying "life" processes apart from the terrible com- plexity of the living organism. The study of isolated enzyme systems has led to important advances in our knowledge and understanding of life processes; yet the student should be reminded that life is more com- plex than its parts. Leibowitz and Hestrin (1945) say: ... it has become clear that the risk involved in translating results from lifeless to living systems is a two-way one: not only may mechanisms which operate in vivo be absent in vitro; mechanisms may be present in vitro and yet not neces- sarily function in vivo. In fermentative physiology, as in biology generally, selective and restrictive activity by the living organism must always be taken into account. The rate of many chemical reactions is changed by the presence of traces of substances which do not appear to enter into permanent chemi- cal combination with the reactants and which appear unchanged when the reaction has come to equilibrium. Substances which alter the rates of chemical reactions are called catalysts, and the process catalysis. Enzymes are catalysts of a very special kind, and many of them catalyze but a single reaction. For example, lactose reacts with water to form glucose and galactose. Unless a catalyst is present, this reaction occurs at a very slow rate. Even at 100°C. a long time is required for an appreci- able amount of lactose to react with water. If, however, some acid is added to the lactose solution, the rate of the reaction is greatly increased, varying in degree with the amount and kind of acid used. This same reaction is catalyzed w^hen the enzyme, lactase, produced by some yeasts and certain other fungi, is added to a solution of lactose. In general, enzymes are specific catalysts. There is no stoichometric relation between the amount of catalyst (acid or enzyme) and the amount of sub- strate decomposed. Within limits, the amount of substrate decomposed per unit of time is dependent upon the amount of catalyst present. For a given set of conditions there is a position of equilibrium where the rate of reaction of the reactants is equal and opposite to the rate of com- bination of the products. The position of equilibrium is not changed by ENZYMES 47 the presence of a catalyst. The same catalyst will effect synthesis as well as decomposition; the position of equilibrium as well as the relative con- centrations of reactants and products determines which reaction pre- dominates. It is possible to choose conditions, in some instances, so the equilibrium conditions favor synthesis. Bourquelot (1915) demonstrated a-methylglucoside was readily formed from methyl alcohol and glucose in the presence of yeast juice. CLASSIFICATION OF ENZYMES It is more important to classify enzymes upon the basis of function rather than the site of action (endo- and exoenzymes). Many enzymes catalyze reactions in which water is either a product (synthesis) or a reactant (degradation). These enzymes are called hydrolases. These reactions usually involve only moderate energy changes. Another class of enzymes, usually intracellular, catalyze oxidation and reduction reac- tions and reactions involving the scission (or formation) of carbon-to-car- bon linkages. These enzymes are known as desmolyzing enzymes and include oxidases, dehydrogenases, and desmolases. Energy changes involved in these reactions are usually large. For more detailed classifi- cations of enzymes see Gortner (1949) and Sumner and Somers (1947). Since an enzyme acts upon a restricted number of compounds, it is convenient to name enzymes with reference to the substrate acted upon. In general, enzymes are named either by adding the suffix -ase to the name of the substrate or by replacing the final syllable of the name of the substrate by this suffix. The following examples give the substrate fol- lowed by the name of the enzyme: maltose, maltase; lactose, lactase; cellulose, cellulase; starch (amylum), amylase; protein, proteinase; pectin, pectinase. The suffix -ase is also used to designate classes of enzymes. Thus, esterases are members of that group of enzymes which catalyze the hydrolysis and synthesis of esters; oxidases are enzymes which activate oxygen, and dehydrogenases are enzymes which activate the hydrogen of various metabolites. An enzyme may have several names. The enzyme which catalyzes the hydrolysis of sucrose is known also as saccharase and invertase. Amylase is also called diastase. Hydrolases. The hydrolases catalyze a wide variety of reactions in which water is either a reactant or a product. Hydrolysis is generally thought of as a process whereby complex molecules react with water to form simpler substances. Many hydrolases are exoenzymes which func- tion by preparing the substrate for assimilation. Among these the follow- ing should be noted : cellulase, amylase, pectinase, various disaccharidases, proteinases, and peptidases. Others are endoenzymes (the same enzymes in some instances), which catalyze the same or similar reactions within the cells. It would be expected that the process of synthesis within the cells 48 PHYSIOLOGY OF THE FUXGI would be of much more common occurrence than outside the cells. In the medium the process of degradation may be expected to go more or less to completion, since the soluble products of the reaction are assimilated by the organism and hence equilibrium is not reached. Within the cell, however, the reverse may be true. Here, the products of hydrolysis may accumulate, a situation which would tend to favor the reverse reaction, or synthesis. Therefore, synthesis within the cell would be expected to occur when a plentiful supply of simple metabolite molecules continue to reach the cell. When few, if any, metabolite molecules are entering the cell, the hydrolysis of reserve materials would take place. These prod- ucts of hydrolysis within the cell are then used in other metabolic processes until the store of reserve material is exhausted. Some of these functions are illustrated in scheme I. Scheme I. General Scheme of Starch Utilization Outside the cell Starch ->- Maltose *- Glucose amylase ^^■^^ ^-^^ maltase Fungus cells many enzymes Carbon dioxide, alcohol and other products of anaerobic respiration Within the cell Glucose -<- many enzymes Carbon dioxide, water and other products of aerobic respiration Glycogen, or other storage products Esterases. These enzymes catalyze the hydrolysis of esters, an acid and an alcohol being formed. The most important natural esters are the fats, which are the glycerol esters of the long-chain fatty acids. Enzymes which catalyze the hydrolysis of fats are called lipases. Both exo- and ENZYMES 49 endolipases are known. Many fungi store fat as reserve material, and presumably the first step in utilization is hydrolysis. Phosphatases are classified as esterases because of the fact that they catalyze the hydrolysis of esters of phosphoric acid. Phosphorus is an essential element which enters into many metabolic processes and is a constituent of many physiologically important compounds. Many coenzymes are esters of pyrophosphoric acid (thiamine pyrophosphate, and diphosphopyridine nucleotide, DPN), while triphosphoric acid is a constituent of triphosphopyridine nucleotide, TPN. The synthetic capacity of the phosphatases has been rarely demonstrated. Other enzymes, phosphorylases, are apparently the catalytic agents active in forming many phosphate esters. In many instances the substrates from which these esters are formed are different from the products of phos- phatase hydrolysis. Carbohydrases. The enzymes which catalyze the hydrolysis of com- plex carbohydrates, or polysaccharides, are called carbohydrases. These enzymes appear to be highly specific ; thus each of the common disaccha- rides requires a different enzyme for hydrolysis. Sucrase is found in many fungi, including the common strains of Saccharomyces cerevisiae, although it is apparently absent in Schizosaccharomyces octosporus. The enzyme which hydrolyzes maltose to glucose is called maltase. Maltase is very widely distributed among the fungi. The enzyme which catalyzes the hydrolysis of lactose to glucose and galactose is called lactase. While this enzyme is less widely distributed among the fungi than sucrase and maltase, it is produced by many species. While it is doubtless correct to assume that the more complex and in- soluble carbohydrates must be hiydrolyzed before utilization, this assump- tion may, in some instances, be false with regard to the disaccharides. It is possible that some fungi may employ a phosphorylative degradation of the disaccharides rather than hydrolysis. For a critical review of carbo- hydrate utilization without preliminary hydrolysis, see Hestrin (1948). In addition to the water-soluble polysaccharides there is a wide variety of water-insoluble high-molecular-weight carbohydrates which are utilized by many fungi as carbon sources. Only two of these complex polysaccha- rides will be considered here. The empirical formula for cellulose is (C6Hio05)„. On complete hydrolysis by acids, glucose is the only prod- uct. Less complete hydrolysis produces a disaccharide known as cello- biose. The majority of fungi, according to Norman and Fuller (1942), are able to attack cellulose. The early work is reviewed by Thaysen and Bunker (1927). With respect to the fungi which attack cellulose, a great deal of variation in cellulolytic ability is found (see White et al., 1948). The enzyme which catalyzes the hydrolysis of cellulose is called cellulase. While starch has the same empirical formula as cellulose, it is more 50 PHYSIOLOGY OF THE FUNGI easily hydrolyzed. Glucose is likewise the end product of hydrolysis. The enzyme (or enzymes) which catalyzes the hydrolysis of starch is called amylase. In general, the end product of enzymatic hydrolysis of starch is maltose and glucose. The various intermediate degradation products are called dextrins. Starch appears to be composed of two main types of compounds: amylose (20 to 25 per cent) and amylopectin. Amylose appears to con- sist of long, unbranched molecules containing some 300 glucose residues, whereas amylopectin has a branched structure. There are two types of amylase: jS-amylase, which hydrolyzes off two glucose residues at a time to form maltose, and oi-amylase, which attacks the 1,4-glucosidic linkages in such a way as to produce starch fragments (dextrins) as the primary products. The dextrins are further hydrolyzed to form maltose and some glucose. The primary function of a-amylase is thus liquefaction; that of the /3-amylase is saccharification. The Aspergillus amylases are of the alpha type. The student is referred to the excellent reviews of Hopkins (1946) and Myrback (1948) for critical summaries of amylase activity. Amylase is widely distributed among the fungi but is not universal. Pectinase. The pectins are colloidal carbohydrate-like compounds found in fruits and in the middle lamellae of plants. Many fungi produce pectinase, which catalyzes the hydrolysis of pectin. When the pectin is hydrolyzed, the cells fall apart. Harter and Weimer (1921) tested the ability of nine species of Rhizoyus to produce pectinase in culture but were unable to correlate the pathenogenicity of these species with the amount of pectinase secreted. In fact, some of the pathogenic species {R. nigricans and R. autocarpi) secreted less pectinase than did two non- pathogenic species {R. chinensis and R. microsporus) . Pectins were formerly believed to yield a considerable variety of hydro- lytic products, including acetic acid, galactose, and arabinose in addition to methyl alcohol and D-galacturonic acid. More recent work indicates that pectins are methylated polymers of D-galacturonic acid (Schneider and Bock, 1937). The chemistry and physiology of the pectins have been reviewed by Bonner (1936). Proteinases and peptidases. These enzymes, also called proteolytic enzymes, catalyze the hydrolysis (and synthesis) of proteins and peptides. These enzymes have been separated into two groups upon the basis of ability to attack native protein. Those enzymes which act upon intact proteins are called proteinases, while those which attack peptides are called peptidases. It seems that the fundamental difference between these two classes of enzymes lies in the point of attack. The proteinases attack the protein molecule in such a way as to produce various peptides as well as amino acids, while the peptidases act only on the ends of the peptide chains. This is analogous to the action of the two amylases. ENZYMES 51 The proteolytic enzymes are a very complex group of hydrolases. In view of the complexity of protein structure this is not unexpected. The question of specificity of the proteolytic enzymes has been considered by Bergmann (1942), who emphasizes that the specificity of a given enzyme for a certain substrate may be modified by the presence of a second sub- strate. Johnson and Berger (1942) have reviewed the enzymatic proper- ties of the peptidases, including those produced by the fungi. Oxidases, hydrogenases, and desmolases. One of the central prob- lems in metabolic processes is how and by what means oxidation of metabolites to carbon dioxide and water is brought about. Some organisms (bacteria) are inhibited or killed by free oxygen (anaerobes). Others may live either in the presence or absence of free oxygen (faculta- tive anaerobes), while others require free oxygen (aerobes) to carry on their metabohc processes and to maintain life. Thus, one organism may degrade a substrate only partially, and these intermediate oxidation products become substrates for other organisms. In the end complete oxidation takes place. In other instances an organism may first carry out a partial degradation and complete it later. Thus, yeast produces alcohol by fermentation. In the presence of oxygen, alcohol is utilized for the synthesis of cellular constituents and as a source of energy. Many fungi possess two ways of obtaining energy by the degradation of metabo- lites: an anaerobic (fermentative) and an aerobic (oxidative) pathway. Both may function in the same organism at the same time, although external conditions may favor one process at the expense of the other, or a substance may inhibit one without affecting the other. Biological oxidations are carried out in two ways: by the removal of hydrogen from, or by the addition of oxygen to, substrates. The name of Wieland is associated with the process of dehydrogenation, and that of Warburg with the second process. The theory of Wieland stressed the importance of the enzyme systems which activated hydrogen or removed hydrogen from substrate molecules, while Warburg's theory focused attention upon the enzyme systems which activated oxygen and which carried oxygen to the substrates. These two theories might seem irreconcilable, but today they are con- sidered as mutually complementary. Both types of enzymatic oxidation are known for the same organism. For further discussion of this problem the student is referred to Elvehjem and Wilson (1944) and Meyerhof et al. (1942) . For a classification of the respiratory enzymes see Gortner (1949) and Sumner and Somers (1947). For the electronic mechanism involved in biological oxidation-reduction see Michaelis (1946). Some representative dehydrogenases and oxidases are aerobic dehj^dro- genases (xanthine oxidase, and uricase); anaerobic dehydrogenases, (succinic dehydrogenase, glucose dehydrogenase, triose phosphate dehy- 52 PHYSIOLOGY OF THE FUNGI drogenase) ; oxidases (cytochrome oxidase, tyrosinase, polyphenol oxi- dase). Succinic acid dehydrogenase oxidizes succinic acid to fumaric acid by the removal of two hydrogens ; but this reaction takes place only in the presence of another system (cytochromes) which "carries" the hydrogen to an oxidizing enzyme, which converts the hydrogen to water and regenerates the cytochrome system so that it can transport more hydrogen. In the cell, succinic acid dehydrogenase is said to be cyto- chrome-linked. In the laboratory, hydrogen carriers other than cyto- chrome may be used. Various other dehydrogenases are linked to the cytochrome system. Another oxidase, tyrosinase, is found in many fungi. It is well estab- lished that copper is an essential constituent of this enzyme system (Kubowitz, 1937) and may be removed by dialyzing the enzyme against cyanide solutions. The activity which is lost by this treatment is restored by cupric ion, Cu++, but other divalent metals do not replace copper. Various reagents which react with copper, such as cyanide, diethyl dithiocarbamate, salicylaldoxine, and carbon monoxide, inhibit the action of tyrosinase. Among the fungi which produce tyrosinase are the follow- ing species (Nelson and Dawson, 1944) : Boletus luridis, Russula foetens, R. niger, Lactarius piperatus, and PsalUota campestris. It is probable that the darkening and coloration of the fruit bodies of these fungi depend upon the activity of tyrosinase. Pyruvic acid, CHs — CO — COOH, is a key compound in carbohydrate utilization, and perhaps in other metabolic processes as well. The enzyme, carboxylase, catalyzes the decomposition of pyruvic acid in the following way: carboxylase CHs— CO— COOH > CO. + CHs— CHO Pyruvic acid Carbon dioxide Acetaldehyde The carbon dioxide formed escapes, while the acetaldehyde formed may be either oxidized to acetic acid or reduced to ethyl alcohol. The enzyme which catalyzes the decarboxylation of pyruvic acid to carbon dioxide and acetaldehyde is abundant in yeast and other fungi. This enzyme con- sists of three moieties, a specific protein, a magnesium ion, and thiamine pyrophosphate. CHEMICAL NATURE OF ENZYMES In the past there has been a great deal of controversy over the chemical nature of enzymes. Sumner (1926) was the first to isolate an enzyme (urease) in pure crystalline condition. Since then a dozen or more enzymes have been prepared in pure crystalline form. All the enzymes which have been isolated in pure crystalline condition have proved to be proteins. ENZYMES 53 Some enzymes are specific proteins requiring neither coenzymes nor metals for activity. These enzymes must contain as an integral part of their structure the specific groups whereby they react with the substrate. Other enzymes consist of two moieties, a specific protein and a specific nonprotein compound which can be detached from the protein. In the process of purifying an enzyme by dialysis the activity may be lost and later restored by adding to the dialyzed material some boiled juice from the tissue under investigation. These specific nonprotein compounds are known as coenzymes. Neither the specific protein nor the coenzyme alone functions as the enzyme; both are required for activity. The specific protein is called the apoenzyme, while the combination of apoenzyme and coenzyme is called the holoenzyme. Still other holoenzymes consist of an apoenzyme, a coenzyme, and a metallic ion. Coenzymes, being non- protein in nature, have proved to be more easily isolated and studied than the specific protein moieties of enzymes. Coenzymes are a varied group of compounds, some relatively simple in structure and others more com- plex. The vitamins are known to enter into the structure of some coenzymes, and it is supposed it is through such coenzyme molecules that the vitamins exert their specific effects. The same coenzyme may com- bine with many specific proteins to form different enzymes. FACTORS AFFECTING ENZYME ACTIVITY Some of the factors influencing enzyme activity affect the intact organ- ism as well as isolated enzyme systems. While the situation within the intact organism is more complex, a knowledge of the behavior of isolated systems will be useful in interpreting the behavior of living fungi. The factors which will be discussed are temperature, hydrogen-ion concentra- tion (pH), chemical reagents (activators and inhibitors), and radiation. Temperature. The rate of many reactions is approximately doubled for each 10°C. increase in temperature. The rate of reactions catalyzed by enzymes also increases with temperature. This increase is not main- tained indefinitely, for enzymes are destroyed by temperatures of less than 100°C. Although there are some reports in the literature of the rate of enzymatic reactions being increased as much as fivefold by a 10°C. increase in temperature, for most enzymatic reactions the increase in rate is less than twofold. This increase between two temperatures 10°C. apart is called the temperature coefficient, or Qio. Since the increase in rate is not exactly constant, it is desirable to specify the temperatures involved; e.g., Q20-30. A reaction with a Qio of 2 proceeds sixteen times faster at 40°C. than at 0°C. Or, the transformation of a given amount of substrate which requires 16 hr. at 0°C. will occur within 1 hr. at 40°C. Figure 9 shows the theoretical effect of temperature upon the amount of substrate trans- 54 PHYSIOLOGY OF THE FUNGI formed when Qio is 2, 3, and 4. It was assumed that one unit of substrate was transformed per unit of time at 0°C. For a reaction with a Qio of 2 an increase in temperature from 28 to 30°C. causes as great an increase in the amount of substrate transformed as does the increase from 0 to 10°C. A small increase in temperature in the range 25 to 35°C. has a greater effect on the rate of reaction than a much greater increase in temperature in the lower temperature range. 80 70 E O60 C QJ '^SO OJ E _o £40 o a> I 30 "in 3 o 20 trt c 3 10 0 1 1 0,0=4 - 7 / J 1 0,0=3-- J / / / / / / 0,0^2-^ ^ _^ y^ ^ 10 20 30 Temperature in degrees centjgrode 40 I' ;g. 9. The theoretical effect of temperature on the rate of enzymatic reactions for different assumed values of Qio. Enzymes are inactivated by heat. The inactivation may be reversible or irreversible depending upon the enzyme involved, the duration of heat- ing, and other factors. The temperature at which the increased rate of reaction is balanced by destruction of an isolated enzyme is the so-called optimum temperature (Bayliss, 1925). The life processes of a fungus are mediated by a large number of en- zymes, which differ in their sensitivity to heat. Fungi cease to grow or reproduce at temperatures lower than that required to kill them. It may be assumed that the enzymes most sensitive to heat are gradually inac- tivated as the temperature increases. This situation in the li^'ing fungus is different from that of an isolated system in that the enzyme is in its natural surroundings and the fungus is able to synthesize or repair the vital enzymes in question. At some temperature we may suppose that the rate of synthesis or repair of the enzyme system is exceeded by the rate of inactivation. When this temperature is reached, or exceeded, the activity of these enzyme systems decrease. This decreased activity is ENZYMES 55 reflected in a lowered rate of growth or may be seen in other behavior of the fungus. With further increases in temperature, the enzyme systems become less and less operative. So long as the temperature does not exceed the point which produces irreversible inactivation, lowering the temperature will enable the fungus to resume growth or other activity. The temperature of inactivation is not fixed unless the length of exposure is also considered. The effect of temperature upon growth is shown in Figs. 5 and 39. The portions of the curves in the optimum temperature range represent a balance between inactivation and increased rate of reaction. Above optimum temperature, the rate of growth falls off abruptly. In a general way the rate of growth parallels that expected of enzymatic processes. Hydrogen-ion concentration. Long ago it was recognized that strong acids and alkalies were destructive to enzymes. A second effect was also recognized: some enzj^mes exhibited maximum activity only in the pres- ence of weakly acidic or alkaline solutions (see Chap. 8 for a discussion of pH). The effect of pH on the activity of urease is shown in Fig. 25. It should be noted that the pH optimum is dependent upon the concen- tration of urea. Haldane (1930) compiled the pH optima of 105 enzymes and found that the range extended from pH 2 to 10. However, all but nine of these enzymes had pH optima between 4 and 8. Most fungi grow between these limits. The effect of the pH of the medium upon the pH of the cell contents is unknown in most instances. Biinning (1936) has reported that the internal pH of the cells of Aspergillus niger is influenced by the pH of the medium. The activities of the exoenzymes are affected by the pH of the medium. Chemical reagents. Some enzymes are inactive or nearly so until they have been treated with certain reagents. A group of the plant proteinases which includes papain and bromelin are activated by hydrogen sulfide and hydrogen cyanide (inhibitors for many enzymes), glutathione, and other thiol compounds. These various activators do not act by removing heavy metals (inactivators for many enzymes) but by reducing the disul- fide linkage, — S — S — , to thiol (sulf hydril) , — SH. Neutral salts activate some enzymes (emulsin, pancreatic amylase). The mode of activation by neutral salts is unknown. Many of the metallic ions (Mg++, Ca++, Fe++, Cu++, Mn++) are required for enzyme activity, but it seems better to consider them as essential parts of some enzymes rather than activators. Inhibitors are substances which reduce or destroy enzyme activity. Inhibition may be reversible or irreversible. A few enzyme inhibitors are cyanides, monoiodoacetate, fluoride, and the hea\^ metals (lead, copper, mercury, silver, etc.). An inhibitor is active against certain enzymes and 50 PHYSIOLOGY OF THE FUNGI not others. There appears to be a close relation between the chemical constitution of the prosthetic group of the enzyme and the inhibitors which inactivate it. We may postulate that inactivation results from a chemical reaction between the inhibitor and the prosthetic group of an enzyme. One characteristic of an oxidase is inhibition by cyanide and hydrogen sulfide. This points to some common moiety in these enzymes which is /Vo cyanide 0.95 X 10'^ M NaCN o — '-' — o- 2.5xlO'^M NaCN U P — o — o- — o — o, l2.4xlO'^MNaCN 50 75 100 Oxygen tension (mm Hg) Fig. 10. The effect of cyanide on yeast respiration. (Courtesy of Winzler, Jour. Cellular Comp. Physiol. 21: 238, 1943. Published by permission of Wistar Institute of Anatomy and Biology.) able to react with cyanide. The oxidases are metalloproteins, and in view of the property of cyanides of reacting with metals to form complexes, it would appear likely that cyanide reacts with the metal to form inactive little-ionized compounds. The typical properties of ferrous and ferric ions are masked by cyanide. Tyrosinase, a copper-containing enzyme, is inactivated by cyanide. Hydrogen sulfide acts on many of the same enzymes which are inhibited by cyanide ; the action may be assumed to be due to the formation of insoluble metal compounds rather than the forma- tion of non-ionized complexes. Winzler (1943) studied the effect of different concentrations of cyanide upon the respiration of yeast maintained under different oxygen tensions. The effect of cyanide on yeast respiration is shown in Fig. 10. It may be ENZYMES 57 noted that tlie percentages of inhibition of respiration (oxygen uptake) depend upon two conditions, the amount of oxygen available and the concentration of cyanide present. We may assume that the cyanide inhibited one or more respiratory enzymes and that, as the concentration of cyanide increased, more and more of these enzymes were inactivated. When the oxygen tension was reduced, these effects were increased. While it is kno^^^l that salts of the heavy metals may denature proteins, and this explanation has been advanced to account for enzyme inactiva- tion by them, recent opinion inclines to the \'iew that the heavy metals inactivate enzymes either by combining with — SH groups, or, under alkaline conditions, by oxidizing thiol sulfur to disulfide. Mercuric ions, especially, may combine with specific metabolites which contain — SH groups (glutathione, thioamino acids), as found by Fildes (1940). Cer- tain metals may inactivate enzymes by replacing the normal metal, ren- dering the enzyme inoperative. It is noteworthy that many enzymes which are inactivated by heavy metals may be either "protected" or restored to activity by the addition of thiol compounds. We may assume for the purpose of illustration that, when a heavy metal combines with an enzyme, an inactive complex or compound is formed as shown in scheme II. Two factors would influence the effectiveness of thiol compounds in preventing or reversing enzyme inactivation, the relative affinity of the enzyme — SH groups and the thiol compound for mercury, and the rela- tive concentration of enzyme and thiol compound. Scheme II. A Scheme Illustrating a Possible Mechanism of Inactivation OF A Sulfhydril Enzyme by Mercuric Ion and Reactr'ation of the Inactive Enzyme-Mercury Complex by the Addition of a Thiol Compound Inactivation Enzyme — S 2(Enzyme— SH) + Hg++^ Hg + 2H+ Enzyme — S Active enzyme Inactive enzyme complex Reactivation Enzyme— S RS \ \ Hg + 2RSH^ 2 (Enzyme— SH) + Hg Enzyme— S RS Active enzyme Radiation. Many reports are to be found in the literature that radia- tion affects enzymes adversely (see the review of Schomer, 1936). Radia- tion may affect not only the enzymes of an organism but also the sub- strates. Ionizing short-wave radiations may cause the formation of hydrogen peroxide from water. Barron et al. (1947) were able, by adding 58 PHYSIOLOGY OF THE FUNGI glutathione, to reactivate phosphoglyceral dehydrogenase which had been inactivated by X rays. Whether radiation is absorbed or not depends upon the chemical con- stitution of the absorbing molecule and the wave length of the radiation. The energy thus obtained may disrupt the molecule or may merely increase its ability to react. These generalizations are not very helpful in either predicting the effect of light upon living fungi or interpreting the observed effects of light on growth and reproduction. It is probable that light acts on various enzyme systems. Light is known to affect one specific enzyme system (cytochrome-cytochrome oxidase). Warburg (1926) showed that the respiration of baker's yeast was inhibited to the extent of 70 per cent in the dark when exposed to carbon monoxide con- taining 5 per cent oxygen, while respiration was inhibited only 14 per cent in light. The same effect of light on carbon monoxide inhil)ition of respiration has been demonstrated with larvae of Tenehris molitor and the heart of embryo trout. Ultraviolet radiation and X rays have a lethal effect on fungi. A small percentagr^ of the spores which survive exposure to ultraviolet radiation may produce mutants. It has been noted recently (Kelner, 1949) that the lethal effect of ultraviolet radiation upon spores of Strep- tomyces griseus is overcome to a considera- ble extent by exposing irradiated spores to visible light. Whether this is due to o o o "" o r^ O ^ Q o o / ^ ^ \p o o/ V€ ° ( Enzyme V ° molecule o ° V Jo o \ /r^ O o o o o o o 0 o Fig. 11. Diagrammatic illustra- tion of the mechanism of enzy- matic hydrolysis. The substrate molecules are represented by small circles, the products of hydrolysis by semicircles. (Cour- tesy of Van Slyke, Advances in Enzymol. 2 : 38, 1942. Published by permission of Interscience Publishers, Inc.) reactivation of certain enzyme systems is not known. MECHANISM OF ENZYME ACTION The most generally accepted theory of enzyme action postulates that the enzyme and substrate unite to form a molecular compound or complex (enzyme-substrate complex). In favorable instances the ex- istence of such enzyme-substrate complexes has been demonstrated (Stern, 1936) . During this temporary union the substrate molecule is " strained" or activated so that it undergoes reaction. The products of the reaction have less affinity for the enzyme surface than the substrate molecules and hence diffuse away, and other substrate molecules unite temporarly with the enzyme and the process continues. If the product molecules are present in excess, they may compete more successfully for the enzyme surface than the substrate does. During synthesis, when the reactants ENZYMES 59 (products) are present in solution in greater than equilibrium concentra- tions, the reactants combine with the enzyme, unite, and diffuse away. Figure 11 gives a diagram which is helpful in visualizing these processes. ADAPTIVE ENZYMES Some fungi produce certain enzymes only in response to particular environmental conditions. Such enzymes are called adaptive enzymes. \\^ether they are produced under all cultural conditions, but in such small amounts as to be undetectable, or whether they are produced de novo is questionable. How^ever, this phenomenon is of great importance. Two types of behavior may be noted when a fungus is placed upon unsuitable medium for the first time. Either the fungus may die, owing to lack of ability to synthesize the enzymes to cope with the new environment; or 240 160 co^ 80 Strain CI -d^ ^-0- o -Aerobic adaptation • -Anaerobic adaptation ■4-1-* I * ■ _1_ ./ 8 16 32 40 24 Hours Fig. 12. Rate of adaptation of a strain of Saccharomyces carlsbergensis to galactose under aerobic and anaerobic conditions. (Courtesy of Spiegelman, Jour. Cellular Comp. Physiol. 25: 128, 1945. Published by permission of Wistar Institute of Anatomy and Biology.) after a time it may synthesize the necessary enzymes, and the fungus is then able to grow and function under the new surroundings. Wlrether or not the fungus is able to synthesize "new" enzymes depends upon its genetic constitution. The biochemical and physiological responses of an organism may change when it is placed on a different kind of medium. These changes ordinarily are called forth by deficiencies in the medium. The substrate upon which the inoculum grew- may be very important in governing the various responses of the organism. Spiegelman (1945) has shown that the adaptation of yeasts to galactose is affected by aerobic and anaerobic conditions. Adaptation is more rapid in air than in nitrogen, and some strains of yeast are unable to adapt to galactose in the absence of oxygen. Figure 12 shows that only some 30 min. is required for Saccharomyces carlshergensis to begin to utilize galactose under aerobic conditions, while about 20 hr. are required under anaerobic conditions. The effect of composition of the medium on the readaptation of panto- ()0 PHYSIOLOGY OF THE FUNGI thenate-dependent strains of yeast to the synthesis of pantothenate has been studied in some detail (Lindegren and Rant, 1947; Lindegren, 1949). Changes to pantothenate independence occurred by an adaptation, which was transmitted vegetatively, and by a gene mutation. The adaptation occurred only in the media of low pantothenate content, while the muta- tions were apparently not affected by the concentration of pantothenate. Leonian and Lilly (1943) studied the induced ability of eight strains of Saccharoniyces cerevisiae to synthesize various vitamins for which they were normally deficient. This was accomplished by long "training" in media "free" from various vitamins. The ability of various yeast strains to synthesize a given vitamin varied. These yeasts which had been trained "reverted" to their deficient status when cultured for 6 months on media containing vitamins and yeast extract. ENERGY AND ENERGY UTILIZATION BY FUNGI Fungi need energy, as well as certain elements and chemical compounds, for life, growth, and reproduction. Since the life processes of the fungi are controlled by interlocking systems of enzymes, the utilization of energy is also an enzymatic process. The chemical reactions which accompany or underlie life processes may be divided into those which yield energy (exergonic) and those which require energy (endergonic) (Coryell, 1940). The oxidation reactions whereby such substrate mole- cules as glucose are converted into carbon dioxide and alcohol or carbon dioxide and water yield energy, while the reactions involved in the syn- thesis of protoplasm and reserve materials require energy. Let us con- sider an analogy first. When water falls from a higher to a lower level, there is a decrease in energy content, and this decrease in energy content is the same whether the water has passed through a turbine or not. The water that passes over a spillway does no useful work, while the water that turns a turbine makes part of the energy available (as mechanical or electrical power) for doing useful work. The energy given up by the falling water is the same in both cases, but only where the proper mecha- nism is available is any useful work obtained. A similar situation occurs when a fungus oxidizes glucose to water and carbon dioxide. If energy-requiring synthetic reactions are coupled with the degradation reactions, a portion of the available energy becomes useful to the fungus. The remainder of the energy liberated appears as heat, which is unavailable to the fungus for lack of suitable mechanisms to utilize it. Winzler and Baumberger (1938) have investigated the liberation of energy by yeast cells during metabolism. Washed yeast cells were sus- pended in a phosphate buffer containing glucose but no nitrogen. The reaction vessel was placed in an adiabatic calorimeter, and the heat ENZYMES 61 evolved and the amount of oxygen absorbed and of carbon dioxide evolved were measured. In the absence of a nitrogen source the synthesis of protoplasm was avoided. The rate at which heat was evolved was con- stant until all the glucose was consumed (exogenous respiration), after which the rate of heat formation decreased (endogenous respiration) (see Fig. 13). 20 50 60 30 40 Time in minutes Fig. 13. Heat produced from glucose oxidation by yeast in the absence of a nitrogen source; 10, 20, and 30 mg. of glucose were added at zero time in curves I, II, and III, respectively. In all cases, only 26 per cent of the expected amount of heat was evolved before the endogenous respiration rate was resumed. (Courtesy of Winzler and Baumberger, Jour. Cellular Comp. Physiol. 12: 199, 1938. Published by per- mission of Wistar Institute of Anatomy and Biology.) In this experiment the theoretical amount of heat could be calculated for the amounts of glucose used. Only 26 per cent of the theoretical heat was produced before endogenous respiration set in. The volume of oxy- gen used was equal to the volume of carbon dioxide evolved, i.e.j the R.Q. was 1. These data may be interpreted as follows: For every molecule of glucose oxidized to carbon dioxide and water, three molecules were syn- thesized into a carbohydrate, presumably glycogen. When sodium acetate was the substrate, about 59 per cent of the theoretical heat was evolved, but in the presence of dinitrophenol the theoretical amount of heat was evolved. This inhibitor, therefore, blocked the assimilative mechanism but not the oxidative processes. Within recent years it has been discovered that certain phosphate esters may play a very important role in energy transfer. The student is referred to the review of Lipmann (1941) for further information on this subject. The utilization of energy derived from degradation reactions depends upon such energy-yielding reactions being coupled with energy-requiring reactions. Degradation reactions which are not so coupled (blocked) 62 PHYSIOLOGY OF THE FUNGI waste energy in the form of heat which is not iitihzed by the fungi. The efficiency of utiHzation depends upon the substrate utihzcd and upon the nature of the coupled reactions. In any case only a part of the energy available in the sulDstrate does useful chemical work for the fungus utiliz- ing it. The application of these ideas with any rigor requires a sound knowledge of thermodynamics. SUMMARY The chemical reactions which underlie the life processes of fungi and other organisms are initiated by organic catalysts, or enzymes. Enzymes catalyze synthetic as well as degradation reactions and are mediators of energy transfer as well. Enzymes are specific proteins which in some instances require certain metallic ions or organic coenzymes, or both, before they are active. In general, an enzyme controls but a single type of reaction. In living organisms these enzyme-controlled reactions are correlated and integrated to a high degree. Among the external factors which modify the action of enzymes the following are especially important: temperature, hydrogen-ion concentra- tion, concentration of substrate and products, and inhibitors. The effects of these factors on isolated enzymes and intact organisms are much the same. While the role of enzymes in maintaining life processes in fungi and other organisms is well established, the application of this information to living fungi must be made with due caution and the realization that a living organism is more complex than its parts. REFERENCES Barron, E. S. G., S. Dickman, and T. P. Singer: On the inhibition ol enzymes by ionizing radiations, Fed. Proc. 6 : 236, 1947. *Bayliss, W. M.: The Nature of Enzyme Action, 2d ed., Longmans, Roberts and Green, London, 1925. Bergmann, M.: A classification of proteolytic enzymes. Advances in Enzymol. 2: 49-68, 1942. Bonner, J.: The chemistry and physiology of the pectins, Botan. Rev. 2: 475-497, 1936. Bourqxtelot, E.: La Synthese biochimique des d-glucosides d'alcools monovalents. II. AlcooW-glucosides a, Ann. chim., Ser. IX, 3: 287-337, 1915. Buchner, E.: Alcoholische Garung ohne Hefezellen, Ber. d. deut. chem. Ges. 30: 117-124, 1897. BtJNNiNG, E.: Ueber die Farbstoff- und Nitrataufnahme bei Aspergillus niger, Flora 131:87-112, 1936. Coryell, C. D.: The proposed terms "exergonic" and "endergonic" for thermo- dynamics. Science 92 : 380, 1940. DrxoN, M.: Multi-enzyme Systems, Cambridge University Press, New York, 1949. Elvehjem, C. a., and P. W. Wilson (Editors): Respiratory Enzymes, Burgess Publishing Co., Minneapolis, 1944. ENZYMES 63 IiLDES, P.: The mechanism of the anti-bacterial action of mercury, Brit. Jour. Exptl. Path. 21: G7-73, 19-40. GoRTNER, R. A.: Outlines of Biochemistry, 3d ed., John Wiley & Sons, Inc., New York, 1949. Haldane, J. B. S.: Enzymes, Longmans, Roberts and Green, London, 1930. Harden, A.: Alcoholic Fermentation, 4th ed., Longmans, Roberts and Green, London, 1932. Harter, L. L., and J. L. Weimer: A comparison of the pectinase produced by different species of Rhizopus, Jour. Agr. Research 22: 371-377, 1921. Hestrin, S.: The fermentation of disaccharides. I. Reducing disaccharides and trehalose, Wallerstein Labs. Communs. 11 : 193-206, 1948. Hopkins, R. H.: The action of the amylases. Advances in Enzymol. 6: 389-414, 1946. Johnson, ]\L J., and J. Berger: The enzymatic properties of peptidases. Advances in Enzymol. 2 : 69-92, 1942. Kelner, a.: Effect of visible light on the recovery of Streptomyces griseus conidia from ultraviolet irradiation injury, Proc. Natl. Acad. Sci. U.S. 35: 73-79, 1949. KuBowiTZ, r.: Ueber die chemische Zusammensetzung der Kartoffeloxydase, Biocnem. Zeit. 292 : 221-229, 1937. Leibowitz, J., and S. Hestrin: Alcoholic fermentation of the oligosaccharides, Advances in Enzymol. 5: 87-127, 1945. Leonian, L. H., and V. G. Lilly: Induced autotrophism in yeast. Jour. Bad. 45: 329-339, 1943. Lindegren, C. C.: The Yeast Cell, Its Genetics and Cytology, Educational Pub- lishers, St. Louis, 1949. Lindegren, C. C, and C. Ralt: A direct relationship between pantothenate con- centration and the time required to induce the production of pantothenate- synthesizing ''mutants" in yeasts. Ann. Missouri Botan. Garden 34: 85-93, 1947. Lipmann, F.: Metabolic generation and utilization of phosphate bond energy, Advances in Enzymol. 1: 99-162, 1941. *Meyerhof, O., et al.: Symposium on Respiratory Enzymes, University of Wisconsin Press, Madison, 1942. MicHAELis, L. : Fundamentals of oxidation and reduction in Currents in Biochemical Research (edited by D. E. Green), Interscience Publishers, Inc., New York, 1946. Myrback, K.: The structure of starch, Wallerstein Labs. Communs. 11: 209-218, 1948. Nelson, J. M., and C. R. Dawson: Tyrosinase, Advances in Enzymol. 4: 99-152, 1944. Norman, A. G., and W. H. Fuller: Cellulose decomposition by microorganisms, Advances in Enzymol. 2 : 239-264, 1942. Pasteur, L.: Etudes sur la vin, Librairie F. Savy, Paris, 1875. Schneider, G. G., and H. Bock: Ueber die Konstitution der Pektinstoffe, Ber. d. deut. chem. Ges. 70: 1617-1630, 1937. Schomer, H. a. : The effects of radiation on enzymes in Biological Effects of Radi- ation (edited by B. M. Duggar), McGraw-Hill Book Company, Inc., New York, 1936. *Spiegelman, S. : The effect of anaerobiosis on adaptation to galactose fermentation by yeast cells, Jour. Cellular Comp. Physiol. 25: 121-131, 1945. Stephenson, M.: Bacterial Metabolism, 2d ed., Longmans, Roberts and Green, London, 1939. Stern, K. G.: On the mechanism of enzyme action. A study of the decomposition 64 PHYSIOLOGY OF THE FUNGI of nioiioctliyl hydrogon peroxide by catalase and of an intermediate enzyme- substrate eompound, Jour. Biol. Chem. 114: 473-494, 1936. Sumner, J. B.: The isolation and crystallization of the enzyme urease, Jour. Biol. Chem. 69: 435-441, 1926. *SuMNER, J. B., and G. F. Somers: Chemistry and Methods of Enzymes, Academic Press, Inc., New York, 1947. Thaysen, a. C, and H. J. Bunker: The Microbiology of Cellulose, Hemicelluloses, Pectins and Gums, Oxford University Press, New York, 1927. *Van Slyke, D. D.: The kinetics of hydrolytic enzymes and their bearing on methods for measuring enzyme activity. Advances in Enzymol. 2 : 33-47, 1942. Warburg, O. : Ueber die Wirkung des Kohlenoxyds auf den Stoffwechsel der Hefe, Biochetn. Zeit. 177: 471-486, 1926. White, W. L., R. T. Darby, G. M. Stechert, and K. Sanderson: Assay of cellulo- lytic activity of molds isolated from fabrics and related items exposed in the tropics, Mycologia 40: 34-84, 1948. WiNZLER, R. J. : A comparative study of the effects of cyanide, azide, and carbon monoxide on the respiration of bakers yeast. Jour. Cellular Conip. Physiol. 21 : 229-252, 1943. *WiNZLER, R. J., and J. P. Baumberger: The degradation of energy in the metabo- lism of yeast cells. Jour. Cellular Comp. Physiol. 12: 183-211. 1938. CHAPTER 5 ESSENTIAL METALLIC ELEMENTS The fungi need about 17 elements to supply their nutritional require- ments. These elements are utilized in the form of specific compounds, as ions, and as free elements. Some of the essential elements are required by all fungi. Other elements are required only by certain species. In a general way, the elements required by the fungi are the same ones required by bacteria, green plants, and animals. There are, however, striking differences in the essential-element requirements of different groups of organisms (Table 15). Differences in ability to utilize specific compounds containing these essential elements are common in the fungi and bacteria. BIOLOGICALLY ESSENTIAL ELEMENTS Before seeking to determine which elements are essential, it is necessary to define what is meant by the term hiologically essential element. An essential element is indispensable in that no other element may entirely replace it. Without these essential elements life is impossible. An ele- ment needed in extremely small amounts may be just as essential as car- bon, which comprises almost half the weight of a fungus. There are some 92 chemical elements (if we exclude the recently isolated trans uranic elements), most of which are known to exist, or may exist, as a mixture of isotopes. So far as is known, all the isotopes of an element (with the possible exception of the isotopes of hydrogen) have the same chemical and biological properties. Even radioactive isotopes, before they decay, exhibit the same biological properties as the stable isotopes. The biological effects of radiation in inducing mutations are considered briefly in Chapter 18. In spite of the limited number of ele- ments, the question of essentiality is not settled completely for all. The problem of determining which elements are essential for the fungi has been approached from the standpoint of ultimate analysis of mycelium and spores. If certain elements, such as carbon, potassium, and mag- nesium, are always found in all samples analyzed, irrespective of the sub- strates upon which these fungi grew, it may be concluded with a high degree of probability that these elements are essential for the fungi. Some of the analytical results of ultimate analyses of mycelium and spores have been collected by Buchanan and Fulmer (1928) and Foster (1949). 65 66 PHYSIOLOGY OF THE FUNGI Organic materials are dried before analysis. On the average about 75 per cent of the fresh weight of mycelium is water, while spores contain only about 40 per cent water. It is probable that the water driven off when fungus cells are dried to constant weight is in part free water and in part water bound to various colloidal cell constituents. Ultimate analyses of mycelium and spores always reveal the presence of carbon and nitrogen. On the average about 45 per cent of dry mycelium is carbon. This high content of carbon makes it certain that carbon is an essential element. The percentage of nitrogen found is quite variable. Phosphorus, potassium, magnesium, calcium, sodium, sulfur, and iron are found in the ash that remains after burning mycelium and spores. More refined methods of analysis reveal that fungus ash contains still other elements. Richards and Troutman (1940) investigated the composition of yeast ash by spectrographic analysis and found the following elements : iron, sodium, boron, bismuth, barium, magnesium, manganese, copper, zinc, tin, lead, tellurium, silver, chromium, potassium, gold, and lan- thanum. However, the mere presence of an element in fungus cells does not necessarily mean that it is essential. Since many of these elements in fungus ash occur in minute traces only, it is desirable to approach the problem of essentiality in another way. This is done by omitting from the medium the element in question. Raulin (1869) was apparently the first to use this method. He found that the omission of phosphorus, sulfur, magnesium, zinc, or iron from the basal medium allowed very little growth of Aspergillus niger. These ele- ments are thus shown to be essential by the two methods of investigation. In general, the experimental work in which specific elements have been omitted from the medium is more convincing than the method of ultimate analysis. This is the preferred method of testing the essentiality of ele- ments required in small amounts. Functions of the essential elements. Thatcher (1934) has attempted to classify the essential elements into groups: structural, functional, and those utilized in the transfer of energy. This classification has some validity and may serve to fix attention upon the more salient biological features of an element. However, most, if not all, of the essential ele- ments play many roles in the life processes of the fungi. In general, the nonmetallic elements may be classified as structural elements. This means that the compounds which make up the structural units such as the protoplasm are largely composed of the nonmetallic essential ele- ments: carbon, nitrogen, hydrogen, oxygen, sulfur, and phosphorus. The functional uses of these elements by the fungi are no less important. The essential metallic elements may be classified as functional elements, but this does not mean that these metallic elements have no structural functions. ESSENTIAL METALLIC ELEMENTS 67 The elements are in the form of chemical compounds, some of which are relatively simple, Avhile others are complex. With the exception of oxygen the essential elements are usually utilized in the form of com- pounds or ions. An essential element may exist in a chemical compound and be unavailable. The properties of a chemical compound are deter- mined by all the atoms that compose it and by the way in which atoms are joined together in the compound. It is convenient to consider the essential elements one by one, but this is done only to simplify the approach to a complex subject. These separate factors must be con- sidered in relation to the organism as a whole. A fungus is no more capable of growth on an iron-free medium than on a carbon- or nitrogen-free medium. Yet, in a balanced medium the ratio of iron to carbon is in the neighborhood of 1 to 50,000. The essential metallic elements function in conjunction with enzyme systems (Chap. 4). This accounts for the small amounts of these elements required. If a vital enzyme system lacks an essential metal ion, it will not function. It appears that in processes such as growth a suboptimal amount of an essential metal will stop growth because the apoenzymes or coenzymes synthesized will lack the necessary activating metal. The ratios as well as the amounts of the various essential metallic ions affect certain metabolic processes other than growth. The absolute amounts of the essential metallic elements required differ widely. Raulin (1869) found that Aspergillus niger required 1 g. of potassium to produce 64 g. of mj^celium, while 1 g. of magnesium sufficed for the synthesis of 200 g. of mycelium. Recent work of Steinberg (1946) with .4. niger indicates still higher yields per gram of these two elements. The yield of mycelium per gram of iron and zinc was in the neighborhood of 55,000 g. The list of metallic elements knoTvni to be essential to fungi has in- creased over the years. The list now includes potassium, magnesium, iron, zinc, copper, calcium, gallium, manganese, molybdenum, vanadium, and scandium. Others will probably be added as cultural methods become more refined and more species are studied. It is unfortunate that only a few fungi have been investigated thoroughly with respect to mineral nutrition. In stating that the above elements are essential, the reserva- tion must be made that they are essential for some fungi under certain conditions. WTiile it may be assumed that all fungi require the same essential elements, experimental evidence is lacking for most species. For the purpose of discussion the essential metallic elements will be divided into two groups, macro and micro metallic elements. This grouping is made solely for convenience and on the basis of the amounts ordinarily employed in culturing fungi under laboratory conditions. 68 PHYSIOLOGY OF THE FUNGI THE ESSENTIAL MACRO ELEMENTS Potassium. This element is essential for all organisms, so far as is known. There is an immense amount of information on the specific effects of potassium on green plants and animals, l)ut such data are not common for the fungi. The quantitative relation between the amount of potassium in the medium and the weight of mycelium produced by Aspergillus niger was studied by Steinberg (1946). This work was done with extraordinary care using a highly purified optimal medium (except potassium). The optimum amount of potassium w-as 150 mg. per liter. The relative amounts of mycelium formed increased as the potassium content of the medium decreased. The fungus produced almost three times as much mycelium per milligram of potassium when 15 instead of 150 mg. per liter were used. Jarvis and Johnson (1950) have reported that Penicillium chrysogenum Q176 requires 40 mg. of potassium and 8 mg. of magnesium per liter of medium for optimum growth. The physiological effects of potassium on fungi have been studied but little. The enzymes in yeast maceration juice which ferment glucose are activated by either potassium or ammonium ions (Muntz, 1947). Mol- liard (1920) noted that a low potassium content of the medium resulted in increased synthesis of oxalic acid by A. mger. The chemical composition of A. niger mycelium varies, depending upon the amount of potassium in the medium (Rippel and Behr, 1934). The problem of biological substitution arose early in the study of fungus nutrition. Biological substitution means that one element can replace another, in whole or in part. The possibility of biological substitution was investigated by Steinberg (1946) using A. niger as the test fungus. This investigation was made to determine whether the alkali metals (lithium, sodium, rubidium, or cesium) could replace potassium, and whether the alkaline-earth metals (calcium, beryllium, strontium, or barium) could replace magnesium. Under these conditions sodium and beryllium gave increased yield of mycelium in media containing sub- optimal amounts of potassium and magnesium. These effects are illustrated in Table 9. Some increases in weight of mycelium were noted under certain con- ditions with some of the other metallic ions tested, but the effects of these elements were ascribed to ion antagonism. Studies of biological substitution require great care and a detailed and extensive knowledge of the composition of the media and of the behavior of the fungus under the experimental conditions used. Magnesium. This element is one of the alkaline-earth group. It is essential for green plants and animals as well as for fungi and bacteria. Aspergillus niger has been more carefully investigated with respect to the ESSENTIAL METALLIC ELEMENTS 69 effects of magnesium than any other fungus. Within certain limits of concentration, the amount of growth of A. niger is proportional to the concentration of magnesium in the medium. This has been demon- strated by Steinberg (1946), Lavollay and Laborey (1938), and others. The application of this principle to the microbiological assay of magne- sium is discussed in Chap. 10. Penicillium glaucum, Botrytis cinerea, and Alternaria tenuis failed to grow in the absence of magnesium (Rabinovitz- Sereni, 1933). Excess magnesium was not harmful to these three fungi until the concentration of magnesium sulfate in the medium reached about 40 per cent. These three species were able to grow in the presence of traces of magnesium but sporulated only when the concentration of mag- nesium was increased. Respiration also increased as the magnesium con- tent of the medium increased. Failure to sporulate unless sufficient magnesium is available is probably to be expected with many fungi. Table 9. The Effect of 50 Milligrams of Sodium on the Amount of Mycelium Produced by Aspergillus niger in an Optimal Medium Containing Twice THE Normal Amounts of INIicro Elements when the Concentration OF Potassium Was Varied (Steinberg, Avi. Jour. Botany 33, 1946.) Potassium, mg. per liter Control, mg. myceUum Sodium added, 50 mg. per liter, mg. mycelium 15 256.3 401.3 30 446.1 783.1 45 641.2 896.7 60 823.4 1,042.0 75 955,2 1,089.0 90 988.0 1,070.0 105 1,065.2 1,093.1 120 1,059.2 1,095.5 135 1,113.9 1,084.9 150 1,145.9 1,146.5 Most of the magnesium in the mycelium of Aspergillus niger can be extracted by means of dilute acids (Ripple and Behr, 1930), which indi- cates that this element does not form stable organic compounds. A rela- tion between the optimum concentrations of magnesium and phosphorus for A. niger was discovered by Laborey et at. (1941). Some 36 phosphate ions are required for every ion of magnesium. Many enzyme sj^stems are activated by magnesium ion, and in view of the role of phosphate in enzymatic transformations it is not surprising that there should be a close relation between magnesium and phosphate concentrations. Magnesium is involved in many of the enzymatic reactions involved in fermentation 70 PHYSIOLOGY OF THE FUNGI (Sumner and Somers, 1947). It is equally likely that magnesium is involved in aerobic oxidation of carbohydrate. Low concentrations of magnesium in the medium led to increased synthesis of riboflavin by A. niger (Lavollay and Laborey, 1938). One ion may affect the physiological action of another. This is called ion antagonism. In nature and in the laboratory fungi come in contact with compounds of both essential and nonessential elements. Many of the nonessential elements are toxic, although toxicity is not limited to the nonessential elements. Copper is an essential element, but it is toxic to most fungi when the concentration exceeds certain limits (Chap. 12). The toxic effect of an ion may be overcome by the presence of one or more other ions in the medium. Gortner (1949) has reviewed this subject from the standpoint of colloidal chemistry and suggests that the relative concentrations of various metallic ions may regulate the process of adsorption. As an example of ion antagonism Lohrmann (1940) described the toxic action of mercuric chloride and boric acid on Aspergillus niger, A. flavus, Mucor pusillus, Penicillium glaucum, Fusarium coeruleum, Cunning- hamella elegans, Ahsidia cylindrospora, and Rhizopus nigricans. The inhibition caused by either of these toxic compounds was overcome in part by increasing the concentration of magnesium sulfate. Similarly, the toxic effects of high concentrations of magnesium sulfate were over- come by mercuric chloride. Either mercuric chloride or boric acid in certain concentrations "stimulated" growth in the nutrient solution used. This is not evidence that either boron or mercury is an essential element, but it does show that the nutrient solution used was unbalanced. The effect of sodium and calcium ions upon growth and respiration of A. niger depended upon the ratio of these nonessential ions present in the medium. A sodium-calcium ratio of 19 to 1 gave the highest rate of respiration, while a ratio of 4 to 1 was most favorable for growth (Gustafson, 1919). Aluminum inhibits the production of itaconic acid by A. terreus. This inhibition is overcome by magnesium sulfate (Lockwood and Reeves, 1945). Nickerson (1946) found the inhibitory effects of zinc ion on the rate of respiration of Epidermophyton floccosum to be reversed by calcium or magnesium ions. The phenomenon of antagonism is not confined to ions. Organic compounds present in media may modify the activity of ions, and organic compounds may antagonize the physiological activity of other organic compounds (Chap. 11). All these possibihties exist. Whether a given ion or compound will be physiologically active depends upon the other constituents of the medium and the metabolic compounds excreted by the fungus under study. ESSENTIAL METALLIC ELEMENTS 71 ESSENTIAL MICRO ELEMENTS These elements have been called heavy-metal nutrients, trace elements, micronutrients, and minor elements. The literature on this subject is extensive and often conflicting. Reviews of this subject are given by Perlman (1949), Foster (1939), and Steinberg (1939). A collection of 10,000 abstracts on the effects of the micro elements on green plants and animals has been published by the Chilean Nitrate Educational Bureau (1948). In spite of Raulin's (1869) discovery that iron and zinc are essential for Aspergillus niger, there arose a school of investigators who considered tho micro elements to be stimulatory rather than essential. This view is no longer held. There are a number of reasons for this misinterpretation: (1) The failure to realize that the "chemically pure" compounds used in preparing media are grossly contaminated from the biological standpoint and that rigorous purification of media is essential in work of this kind. (2) Distilled water is often a source of metallic ions unless it has been redistilled in Pyrex, or preferably quartz, stills. (3) Many kinds of chemical glassware are sufficiently soluble to furnish the fungi all or a part of the micro elements required. (4) The inoculum, whether mycelium or spores, may introduce sufficient micro elements to obscure the need for these elements. Serial transfer using media free from the element in question and the use of small inocula minimizes this source of error. Steinberg (1936) has indicated that the optimum concentration of the essential micro metallic elements for A. niger ranges from 0.3 mg. of iron to 0.02 mg. of gallium per liter of medium. Lest the reader conclude that these concentrations are so small as to be meaningless, it is revealing to calculate the number of atoms of iron in 0.3 mg. From the atomic weight of iron and Avagadro's number it may be calculated that there are about 3 X 10^^ atoms in 0.3 mg. of iron. If the number of cells produced by A. niger under these conditions were known, the number of iron atoms available for each cell could be calculated. In lieu of this information we may use data from experiments on the number of yeast cells produced in a liter of medium. Under favorable conditions there are roughly 500 billion yeast cells produced in a liter of medium (Stark et al., 1941). If A. niger produces the same number of cells per liter as yeast, there would be available 6.4 X 10^ atoms of iron per cell. The prime essential in investigations dealing with the effects of the essential micro elements is a medium free from the element under study. This ideal is difficult to attain in practice. Equal care is necessary in the choice of culture vessels, for it is wasted effort to remove an element from the medium rigorously and then contaminate it by using glassware which 72 PHYSIOLOGY OF THE FUNGI furnishes the metal. The culture A^essels should be of quartz for work of the most exacting kind, although Pyrex or other suitable glassware may be used. It is desirable in any event to use a few quartz culture vessels as controls. In part, the long controversy over the effect of zinc on fungi was due to the liberation of sufficient amounts of this element from certain kinds of glassware used as culture vessels. Javillier (1914) showed that the addition of zinc to cultures of A . niger has little effect when Jena glass culture flasks were used. When quartz vessels were used, the crop increased from 291 mg. in the control without added zinc to 1,624 mg. when zinc was added. Steinberg (1919) found essentially the same results except that zinc deficiency could be demonstrated for A. niger when Pyrex vessels were used (Table 10) . Table 10. The Average Weight of Five Cultures of Aspergillus niger Culti- vated ON the Same Basal Medium in Three Makes of Glassware (Steinberg, Am. Jour. Botany 6, 1919.) Make of glassware Jena Kavalier Bohemian . Pyrex Mg. mycelium Zinc, 10 mg. per liter 987 943 957 Purification of culture media. Progress in the study of essential micro elements depends upon methods of removing them from media. As long as these elements occur in the ingredients of the media, their need may be unnoticed and unsuspected. In 1919 Steinberg devised a useful method of reducing the concentration of heavy metals, especially iron and zinc, in media. In essentials, this method consists in autoclaving the complete medium with 15 g. per liter of calcium carbonate. The hot solution after autoclaving is filtered through paper or a fritted-glass filter, or allowed to cool and the supernatant liquid decanted off. The precipitate must be removed; otherwise the essential elements will be released by the fungi. Calcium oxide and magnesium carbonate may replace calcium carbonate in some applications (Steinberg, 193oa) . The mode of purification appears to be as follows: During autoclaving, heavy-metal carbonates or their hydroxides are formed. The excess calcium carbonate serves to adsorb these insoluble compounds The composition of a medium is somewhat changed by this treatment, part of the phosphate being removed as cal- cium phosphate. In practice this is compensated by using an excess of phosphate. A medium which is treated by this process is essentially neutral in reaction, which may lead to some changes in the sugar during autoclaving. ESSENTIAL METALLIC ELEMENTS 73 Sugars are frequently highly contaminated with metallic compounds. Steinberg (1937) has reported a sample of glucose to contain the following elements: lithium, sodium, strontium, calcium, rubidium, potassium, manganese, aluminum, iron, rhodium, nickel, silver, copper, magnesium, tin, boron, and silicon. The metallic contamination of non-ionic com- pounds such as the sugars can be sharply reduced by a variety of mild procedures. Shu and Johnson (1948) give these details for an aluminum hydroxide coprecipitation method: To 140 g. of glucose contained in 500 ml. of solution, 1.25 g. of Al2(S04)3'18H20 were added. Dilute ammo- nium hydroxide was added until the pH rose to 9, and the precipitate of A1(0H)3 Avith the adsorbed impurities was filtered off. This treatment was repeated until the desired degree of purification was attained. Non- ionic substances such as glucose and urea may be purified by treatment with cation-exchange materials operating on the hydrogen cycle. Perl- man (1945) used Zeo-karb H (Permutit Corporation) for this purpose. Various ion-exchange materials are used to purify beet juice in the manu- facture of sugar. Mulder (1939-1940) found the combination of ammo- nium sulfide and Norit to be efficient in removing copper from media. The sulfide ion forms insoluble heavy-metal sulfides while the activated carbon serves as a "gatherer." Complex-forming reagents such as diphenylthiocarbazone (dithizone) (Stout and Arnon, 1939), and 8-hydroxyquinoline (Waring and Werkman, 1943) are useful in removing heavy-metal ions from, or testing the purity of, salts used in preparing media. These reagents and the metal com- plexes they form are removed from solutions by extraction with chloro- form or other organic solvents. The chemistry of complex formation between organic compounds and ions is treated by Yoe and Sarver (1941). Others have merely added such complex-forming reagents to the media, in which the various metallic ions combine with the reagent to form non- available compounds. The specificity of the reagent, concentrations of reagent and the metallic ion or ions, the pH of the medium, as well as the stability of the complex, enter into the success of this type of treatment. Hickey (1945) found that 2,2'-bipyridine inactivated ferrous iron in media treated with this reagent. It is better to remove metallic impurities from the media by extraction than to depend upon complexing compounds to hold these ions in non-ionic combination. Certain compounds used in making media such as the amino acids and hydroxy acids form non-ionized complexes with various metallic ions. Media containing these types of compounds are difficult to free from metallic contamination. In addition, some fungi excrete hydroxy acids, such as citric acid, which may modify the availability of the essential micro elements. Media may be freed of essential micro elements by a biological process. 74 PHYSIOLOGY OF THE FUNGI If a fungus is grown on a medium, it will absorb and utilize the essential elements present in the medium. The success of this procedure depends upon having a low initial concentration of the essential micro elements, which soon become exhausted so that the culture liquid no longer supports growth. Removal of the mycelium will thus remove the elements which have been taken up. The culture filtrate may then be used as a medium relatively free of micro elements. However, fungi excrete various com- pounds which may affect the results. MacLeod and Snell (1947) have recently utilized this method in studying the mineral nutrition of some lactic acid bacteria. Iron. Raulin's claim that iron was essential for fungi was questioned at first, but his findings were soon confirmed. So far as is known, iron is essential for all fungi. It may be noted that, in the absence of another essential element in the medium, iron alone may cause little or no response. If the zinc content of a medium is low, the addition of iron to an iron-free medium will have little effect. This situation is true of any essential nutrient. Only one element may be studied at a time, but all the other essential nutrients must be present before the effect of the nutrient under investigation can be studied. Some results of Steinberg (1919) with Aspergillus niger on media purified by the calcium carbonate method are given in Table 11. Neither iron nor zinc alone had much effect on the growth of A. niger, since both of these elements are essential for this fungus. Table 11. The Effect of Iron and Zinc, Singly and in Combination, on the Amount of Growth of Aspergillus niger (Steinberg, Am. Jour. Botany 6, 1919.) Essential Micro Element Mg. Mycelium Added Control (none added) 18 Iron 44 Zinc 40 Iron plus Zinc 731 Little interest has been shown in recent years in proving iron to be an essential element for a large number of fungi. In view of the almost uni- versal occurrence of a group of iron-containing enzymes (catalase, the cytochromes, cytochrome oxidase, etc.), the essential role of iron is taken for granted. The most obvious effect of suboptimal iron concentrations upon fungi is decreased growth. This result is probably due to the decreased and limited amounts of iron-containing enzymes formed under these condi- tions. It was shown by Yoshimura (1939-1940) that the amount of catalase produced by Aspergillus oryzae increased as the amount of iron in the medium increased. Lilly and Leonian (1945) showed that a rela- ESSENTIAL METALLIC ELEMENTS to tion existed between the amount of iron supplied in the medium and the ability of Rhizohium trijolii to synthesize certain vitamins. In the pres- ence of suboptimal concentrations of iron the addition of certain vitamins replaced iron to a certain degree. A quantitative study of the ^ntamins synthesized by Torulopsis utilis has shown the iron concentration to be important (Lewis, 1944). Increased amounts of thiamine, riboflavin, nicotinic acid, and pyridoxine were synthesized on media low in iron, while the amounts of biotin, inositol and p-aminobenzoic acid were decreased. L) 10 15 20 Mg. ferric sulfate per liter Fig. 14. The effect of iron [Fe2 (804)3] in overcoming the inhibitory action of copper (CuS04-5H20) on the production of penicilUn by Penicillium chnjsogenum X-1612. An amount of copper sufficient to inhibit peniciUin production entirely did not affect the amount of growth. The fungus was cultured submerged in a lactose-starch- dextrin-ranmonium sulfate medium for 7 days. (Curves drawn from data of Koffler et al., Jour. Bad. 53 : 120, 1947. Pubhshed by permission of The Williams & Wilkins Company.) There has been a great deal of interest in the effects of iron and other metallic ions on various microbiological processes. Perlman et al. (1946) have shown that the iron concentration is an important factor in citric acid fermentation by Aspergillus niger. The optimum iron concentration for citric acid production varied over tenfold for different strains of A. niger. The effect of iron on penicillin production has been studied by Kofl^ler et al. (1947), who concluded that the effect of the ash of corn steep is due to iron and phosphate. Chromium increased penicillin production above that obtained with iron and phosphate, presumably by neutralizing the effect of other ions. Similarly an antagonism was shown to exist between copper and iron. The antagonistic effect of copper and iron on the production of penicillin by Penicillium chrysogenum X-1612 is shoA\Ti in Fig. 14. 76 PHYSIOLOGY OF THE FUNGI The iron concentration of the medium has been shown to affect the amount of pigmentation of Torulopsis pulchcrrima (Roberts, 1946). Zinc. This element is essential for Aspergillus niger (Raulin, 1869; Steinberg, 1919). Foster (1939) lists Trycliophytoninterdigitale, Rhizopus nigricans, and Saccharomyces cerevisiae as recjuiring zinc, and Roberg (1928) found zinc to be essential for A. fumigatus and A. oryzae. Blank (1941) reported the amount of growth oi Phymatotrichum omnivorum to be increased by the addition of zinc to a medium treated with calcium car- bonate, and Perlman (1948) noted that the sclerotia of Sclerotium delphinii are more highly pigmented in the presence of added zinc. Zinc ions activate (and inhibit) various enzymes such as enolase and dipeptidase. Zinc is contained in carbonic anhydrase, an enzyme which catalyzes the decomposition of carbonic acid to carbon dioxide and water. In addition to these specific uses the zinc concentration has a decided effect on a number of physiological or biochemical processes in fungi. Foster and Waksman (1939) found that the production of fumaric acid from glucose by Rhizopus nigricans varied according to the amount of zinc added to the medium. Fumaric acid was produced most efficiently when the concentration of zinc was low (1.2 mg. per liter). Higher concentra- tions of zinc resulted in increased growth and decreased production of fumaric acid. From these results it appears that zinc plays a role in the utilization of glucose, the completeness of oxidation and assimilation being favored by relatively high concentrations of zinc. A somewhat similar effect of zinc on the production of lactic acid by Rhizopus sp. has been noted (Waksman and Foster, 1938). Zinc was found to cause increased growth and a decrease in the production of lactic acid, while the effect of iron is to increase the yield of lactic acid. For a further dis- cussion of the mechanism of zinc in fungus metabolism, see Foster (1949). Copper. This element is essential for animals, green plants, and fungi. From the work of Steinberg (1936) it appears that 0.04 mg. of added copper per liter of purified medium is sufficient for the maximum growth of Aspergillus niger. Under these conditions omission of copper decreased the yield only from 984.8 to 774.3 mg. It is probable that purification of the medium by the calcium carbonate treatment is not very satisfactory for this element. The ^veight of metal needed to obtain maximum growth with A. niger is much less for copper than for iron or zinc. The experi- mental difficulties increase as the amount of a micro element needed becomes less. Apparently it is very difficult to prepare a copper-free medium. Roberg (1931) made use of Bortel's method of adding a trace of ammonium sulfide to convert heavy metals to sulfides and adsorbing these impurities with charcoal. This treatment is very efficient in remov- ing iron and zinc but somewhat less satisfactory for removing copper. The essential nature of copper for A. flavus and Rhizopus nigricans was ESSENTIAL METALLIC ELEMENTS 77 shown by McHargue and Calfee (1931). The full effect of copper was dependent upon the presence of other essential elements. The coloration of conidia of A. niger has been shown to depend upon the copper content of the medium (Javillier, 1939). Although copper is an essential element, it is a constituent of many fungicides (Chap. 12). The concentration, therefore, is a very important consideration in studying the effect of this element. The phenomenon of ion antagonism must also be considered, for the effect of a given amount of copper is dependent upon the other constituents of the medium. Marsh (1945) investigated the antagonistic effects of three salts upon copper as it affected germination of conidia of Sclerotinia fructicola Table 12. The Antagonistic Effect of Three Salts on Copper as Shown by THE Germination of Conidia of Sclerotinia fructicola (Marsh, Phytopathology 35, 1945.) Salt concen- tration Percentage Germination in 4 X 10-^/ CuS04, plus 0.01% glucose MgS04 CaCh KCl 0.0 1.2 0.8 2.9 10-^M 54.0 31.0 3.9 10-^il/ 67.0 62.0 2.6 IQ-'M 78.0 83.0 3.9 10-2M — 59.0 (Table 12). It was shown that the mechanism of the protective action of these salts was to decrease absorption of copper. There is no reason to assume that the absorption and utilization of copper from nutrient solu- tions would not be affected similarly. Thus, the amount of copper added to a nutrient solution may reflect only imperfectly the amount absorbed and used by a fungus. It was noted in Chap. 4 that copper is an essential constituent of certain enzymes, including tyrosinase, which occurs in many fungi. Nelson and Dawson (1944) suggest that tyrosinase functions in the respiration chain as an oxygen shuttle. Manganese. The classification of this element as essential rests upon the experimental findings that omission of this element from media results in decreased yields. The multiplication of examples strengthens the validity of this conclusion, although most investigators have confined their attention to a relatively few species. The results of Robbins and Hervey (1944) with Pythiomorpha gonapodyoides indicate that investiga- tion of fungi other than Aspergillus niger with regard to micro-element 78 I'lIYSlOWGY OF THE FUSGl nutrition may be rewarding. It was unnecessary to resort to elaborate methods of medium purification to demonstrate that manganese is essen- tial for P. gonapodyoides. This situation occurred only when reagent magnesium sulfate of a certain manufacture was used. Substitution of another brand of magnesium sulfate revealed heavy (biological) con- tamination by manganese (Fig. 15). The inoculum was found to carry sufficient manganese and other micro elements to influence the amount of growth in the first passage. No growth resulted in the third passage in A B Fig. 15. Pythiomorpha gonapodyoides growing in a basal solution \Yith no added mineral supplements. A, medium prepared with Baker's Analyzed magnesium sulfate. B, medium prepared with Mallinckrodt's magnesium sulfate analytical reagent. Age, 5 days. Note the extensive white mj'celium in A and the slight growth in B. (Courtesy of Robbins and Hervey, Bull. Torrey Botan. Club 71: 263, 1944.) the absence of added manganese. The range of manganese concentra- tions for optimum growth was narrow and appeared to depend upon the concentration of other micro elements present, particularly zinc. Stein- berg (1935) found manganese to be essential for A. niger. McHargue and Calfee (1931, 1931a) noted that growth of A. flainis, Rhizopus nigricans, and Saccharomyces cerevisiae increased in the presence of added manganese. Steinberg (1945) showed that omission of manganese from a balanced medium resulted in a decrease in yield of A. niger from 1,084.8 to 356.6 mg. No spores formed when manganese was omitted. It is interesting to note that, as the numbers of spores used for inoculum ESSENTIAL METALLIC ELEMENTS 79 decreased, A. niger became more sensitive to micro-element deficiencies in the medium. The favorable effect of adding biotin to the medium when only a few spores were used as inoculum suggests an intimate con- nection between micro-element nutrition and the synthesis of this vita- min. Whether the decreased yield due to small inoculum was due to other deficiencies or to a decreased rate of growth is not entirely clear, as all harvests were made after 4 days. Manganese (Mn++) has been shown to be the natural activator of yeast arginase. Other enzymes are activated by this element (Sumner and Somers, 1947). In view of the small amounts of manganese required by fungi, it may be assumed that manganese functions as a constituent of various enzymes. Molybdenum. The study of the role of this element emphasizes the similarity in certain physiological processes throughout the plant king- dom. The most striking feature of this essential element is its role in nitrogen metabolism. The utilization of nitrate nitrogen by green plants and fungi and the fixation of atmospheric nitrogen by bacteria {Azotohac- terchroococcum, Clostridium pasteimanum) is dependent upon molybdenum (Bortels, 1930, 1936). Our knowledge of the effect of molybdenum on fungi is largely confined to Aspergillus niger. Steinberg (1936, 1937) found that more molybde- num was required by A. niger for maximum growth in media containing nitrate nitrogen than in media with ammonium nitrogen. Steinberg expressed the opinion that molybdenum is essential for A . niger even when ammonium nitrogen is available. Additional studies on A. niger and other organisms (Mulder, 1948) indicated that an increased need for molybdenum is associated with nitrate utilization. It may be assumed that the enzymatic reduction of nitrate is carried out by enzymes which require molybdenum as an activator. In view of the important role of molj^bdenum in the utilization of nitrate nitrogen, care should be used in comparing the value of different nitrates. Unless sufficient molybdenum is present, misinterpretations may result. Steinberg (1937) found the amount of molybdenum present as an impurity in various nitrates to vary. One sample of calcium nitrate contained enough molybdenum to support maximum growth of A. niger. Perhaps the report of Young and Bennett (1922) that many fungi made better growth on calcium than on potassium nitrate may be partially explained on the basis of the molybdenum content of these two salts. This explanation, of course, must allow for the effect of calcium, which is now known to be essential for certain fungi. Calcium. This element was one of the first to be recognized as essen- tial for green plants and animals. In 1922, Young and Bennett reported that Rhizocionia solani made no growth in the absence of this element 80 PHYSIOLOGY OF THE FUNGI This report apparently attracted little attention since most investigators working on this problem confined their attention to Aspergillus niger. The value of using more than one fungus to demonstrate the essential nature of calcium was strikingly shown by Steinberg (1948, 1950). These data are given in Table 13. It is evident from the data in Table 13 that the essential nature of cal- cium for certain fungi is established. The concentrations of calcium required for maximum growth varied from 2 to G mg. per liter of medium. On the other hand, neither A. niger nor Fusariuni oxysporum needs more Table 13. Effect of the Omission of Calcium from the Medium on the Growth of Seven Fungi (Steinberg, Science 107, 1948.) Fungus Aspergillus niger Rhizoctonia solani Sclerotium I'olfsii Cercos-pora nicotianae Fusarium oxysporum var. nicotianae. Pythium irregulars Thielaviopsis basicola Calcium added, Calcium not added, mg. mycelium percentage of yield 1,250.0 100.0 1,215.1 14.3 1,082.3 49.5 1,380.2 90.1* 823.3 100.0 459.0 60.1* 364.2 82.0* * Asparagine of unknown purity was used as a sovirce of nitrogen. than spectroscopic traces of calcium, if they require this element at all. Steinberg is of the opinion that further advances in purity of nutrient solutions will reveal more uniformity in the essential element require- ments of organisms. Lindeberg (1944) has demonstrated a synergistic effect between manga- nese and calcium upon the growth of various species of Marasmius. Table 14. The Effect of Increasing Concentrations of Calcium and Manga- nese, Alone and in Combination, on the Growth of Marasmius epiphyllus (Lindeberg, Symbolae Botan. Upsalie7isis, 8: 2, 1944.) (Dry weight mycelium in milligrams.) Mn, millimoles per liter Ca, millimoles per liter 0 0.005 0.05 0.5 0.0 0.0005 0.005 0.05 0.5 10.1 11.1 10.7 18.2 20.3 19.8 18.0 18.3 20.8 35.8 33.0 38.4 35.7 47.6 48.6 73.5 83.8 78.5 77.0 52.6 ESSENTIAL METALLIC ELEMENTS 81 Within limits, the growth of M. aUiaceus and M. epiphyllus was propor- tional to the concentration of either of these elements, and the response to each element was modified by the presence of the other. The data in Table 14 illustrate this effect. In addition to the essential micro elements discussed above, there is some evidence which indicates the essentiality of other metallic elements for the fungi. Certain of these elements are essential for other organisms. Gallium. Under certain conditions Steinberg (1938) was able to show that omission of this element from the medium led to decreased yield and sporulation of Aspergillus niger. Extraordinary care was needed to demonstrate gallium deficiency. The chemicals used were spectroscopi- cally pure with the exception of traces of iron, calcium, and sodium. The sucrose, after 6-hr. extraction with alcohol, contained only 0.0014 per cent ash. The water used was triple-distilled, the last distillation being made in a quartz still. Spectroscopically pure calcium oxide w^as used to purify the sucrose further. Under these conditions the yield of A. niger increased from 814 mg. to 1,053 mg. when gallium (0.02 mg. per liter) was added to the medium. The salts of 76 other chemical elements were tested, and none was found to replace gallium. In view of the similar chemical behavior of gallium and aluminum, Steinberg considers it possi- ble that the biologic activity sometimes attributed to aluminum may in reality be due to gallium. Scandium. In the discussion of the role of manganese in nitrogen metabolism it w^as noted that the amount of manganese required was determined by the nitrogen source used. In a somewhat similar fashion, Steinberg (1939) found that scandium appeared to be essential when glycerol was used as a carbon source for Aspergillus niger. Growth was poor on this carbon source ; omission of copper or manganese increased the yield somewhat. Omission of scandium decreased the yield from 269.4 to 107.4 mg. Interestingly enough, scandium appeared to have no effect on growth when sucrose was used as a source of carbon. Addition of lysine or proline (20 mg. per liter) to the glycerol medium increased growth and at the same time prevented the effect of scandium. These results suggest that the need for certain elements may be shoA\Ti only under certain nutritional conditions. Vanadium. Bertrand (1943) reported the presence of this element in all fungi examined. Amanita muscaria contained from 61 to 156 mg. of vanadium per kilogram. Bertrand (1941) considers vanadium as an essential element for Aspergillus niger. Cobalt. Whether fungi require some, or all, of the other metallic ele- ments required by other organisms is not kno^vn. Cobalt is required by animals. Lack of sufficient amounts of this element in the soil causes severe cobalt deficiency in animals which are pastured on such soils. 82 PHYSIOLOGY OF THE FUNGI Recently, a cobalt-containing vitamin (B12) was isolated. This vitamin is synthesized by Streptomyces griseus (Rickes et al., 1948) and some bacteria. Whether S. griseus requires cobalt as an essential element for growth or reproduction is not known. The synthesis of this vitamin is necessarily dependent upon a supply of cobalt. Some bacteria are known to be deficient for vitamin B12. PERIODICITY OF BIOLOGICALLY ESSENTIAL ELEMENTS Steinberg (1938a), Frey-Wyssling (1935), and others have considered the problem of biologically essential elements in relation to the structure Table 15. A Portion of the Periodic Table of Elements Based on Atomic Number The biologically essential elements are set in italics. Those elements essential for fungi are marked with an asterisk. Group Group Group Group Group Group Group Group Group 0 1 2 3 4 5 6 7 8 H* 1 He Li Ce B C* N* 0* F 2 3 4 5 6 7 8 9 Ne Na Mg* Al Si P* S* CI 10 11 12 13 14 15 16 17 A 7v'* Ca* Sc* Ti V* Cr Mn* Fe* Co Ni 18 19 20 21 22 23 24 25 26 27 28 Cu* Z71* Ga* Ge As Se Br 29 30 31 32 33 34 35 Kr Rb Sr Y Zr Cb Mo* Tc Ru Rh Pd 36 37 38 39 40 41 42 43 44 45 46 Ag Cd In Sn Sb Te I 47 48 49 50 51 52 53 and atomic number of the elements. The biologically essential elements are in italics in Table 15. It is noteworthy that the essential elements tend to occur in groups with consecutive atomic numbers. Atomic num- ber is a fundamental property of atoms and denotes the number of excess positive charges on the nucleus. Only those elements which have certain configurations are required by organisms. Why some organisms require certain elements not required by others is not known. ESSENTIAL METALLIC ELEMENTS 83 SUMMARY The role of the essential metallic elements is primarily functional rather than structural. Presumably these ions usually function in ionizable combinations, but some compounds containing metals in non-ionizable compounds have been isolated from fungi. It may be assumed that many of these metallic ions activate enzyme systems, while others are integral parts of enzymes and other essential organic compounds. An element is essential because some of its vital functions cannot be replaced by any other element. Some functions may be performed by other closely related elements. The concentration of an essential element affects many life processes besides growth, which is the usual criterion of essentiality. The concen- trations of various essential ions influence the formation of pigments, the synthesis of vitamins and other products, and the dissimilation of carbo- hydrates. While the essential elements may be supposed to participate uniquely in certain life processes, the concentrations of other ions, both of essential and nonessential elements, modify the action of a given ele- ment. The phenomenon of ion antagonism no doubt exists among all ions, and in evaluating the effects of any element it is necessary to con- sider the other constituents present in the medium. It is probable that the mechanism involved is one of modified adsorption rather than any direct chemical reaction in the medium. The widespread use of Aspergillus niger as a test fungus in micro- element studies has had the advantage that the work in many laboratories may be compared. The careful and long-continued studies by Steinberg are especially valuable. The almost exclusive use of this fungus has also had its disadvantages. Comparatively little is known about the need of other species for micro elements. Other fungi may require some of these elements in amounts which make it comparatively easy to demonstrate deficiency. The evidence for the essentiality of iron, zinc, copper, manganese, molybdenum, and calcium is impressive in most instances, but the need for the elements on the part of all fungi under all cultural conditions has not been established. In a few instances the evidence is confined to a single fungus. The micro-element nutrition of a wide range of species needs further study. REFERENCES Bertrand, D.: Le Vanadium comme facteur de croissance pour V Aspergillus niger, Bull. soc. chim. biol. 23: 467-471, 1941. Bertrand, D.: Le Vanadium chez les champignons et plus sp^cialement chez les Amanites, Bull. soc. chim. biol. 25: 194-197, 1943. Blank, L. M.: Response of Phymatotrichum omnivorum to certain trace elements, Jour. Agr. Research 62: 129-159, 1941. 84 PHYSIOLOGY OF THE FUNGI BoRTELS, IT.: Molybdfln als Katalysator bei der biologischen Stickstoffbindung, Arch. Mikrohiol. 1 : 333-342, 1930. BoRTELS, H. : Weitere Untersucluingen fiber die Bedeutung von IVrol3'bdan Vanadium, Wolfram uiid anderen Erdaschenstoffen fiir stickstoffbindende und andere Mikroorganismen. Cent. Bakt., Abt. II 95: 193-218, 1936. Buchanan, R. E., and E. I. Fulmer: Physiologj^ and Biochemistry of Bacteria, Vol. I, The Williams & Wilkins Company, Baltimore, 1928. Chilean Nitrate Educational Bureau: Bibliography of the Literature on the Minor Elements and Their Relation to Plant and Animal Nutrition, New York, 1948. *Foster, J. W.: The heavy metal nutrition of fungi, Bolan. Rev. 5 : 207-239, 1939. Foster, J. W.: Chemical Activities of Fungi, Academic Press, Inc., New York, 1949. Foster, J. W., and S. A. Waksman: The specific effect of zinc and other heavy metals on growth and fumaric acid production by Rhizopus, Jour. Bad. 37: 599-617, 1939. Frey-Wyssling, A.: Die unentbehrlichen Elemente der Pflanzennjihrung, Natur- wissenschaften 23: 767-769, 1935. GoRTNER, R. H.: Outlines of Biochemistry, 3d ed., John Wiley & Sons, Inc., New York, 1949. GusTAFSON, F. G.: Comparative studies on respiration. IX. The effects of antago- nistic salts on the respiration of Aspergillus niger, Jour. Gen. Physiol. 2: 17-24, 1919. HicKEY, R. J. : The inactivation of iron by 2,2'-bipyridine and its effects on riboflavin synthesis by Clostridium acetobutylicum, Arch. Biochem. 8: 439-447, 1945. Jarvis, F. G., and M. J. Johnson: The mineral nutrition oi Penicillium chrysogenum Q176, Jour. Bad. 59: 51-60, 1950. Javillier, M.: Une cause d'erreur dans I'etude de Taction biologique des elements chimiques: la presence de traces de zinc dans la verre, Compt. rend. acad. sci. 158: 140-143, 1914. Javillier, M.: Cuivre et Aspergillus niger. Rappel de quelques faits anciens, Ann. fermentations 5: 371-381, 1939. *KoFFLER, II., S. G. Knight, and W. C. Frazier: The effect of certain mineral ele- ments on the production of penicillin in shake flasks, Jour. Bad. 53: 115-123, 1947. Laborey, F., J. Lavollay, and J. Neumann: Coefficient d'action du magnesium vis-a-vis d' Aspergillus niger: variation lineaire de ce coefficient avec la concen- tration en phosphore, Compt. rend. acad. sci. 212: 624-626, 1941. Lavollay, J., and F. Laborey: Sur les circonstances d'apparition de pigments jaunes dans le liquide de culture d' Aspergillus niger, Compt. rend. acad. sci. 206 : 1055- 1056, 1938. Lewis, J. C. : Relationship of iron nutrition to the synthesis of vitamins by Torulopsis utilis, Arch. Biochem. 4: 217-228, 1944. Lilly, V. G., and L. H. Leonian: The interrelationship of iron and certain factors in the growth of Rhizohium trifolii. strain 205, Jour. Bad. 50 : 383-395, 1945. Lindeberg, G.: Ueber die Physiologic Ligninabbauender Boden Hymenomyzeten, Symbolae Botan. Upsalienses 8(2): 1-183, 1944. LocKwooD, L. B., and M. D. Reeves: Some factors affecting the production of itaconic acid by Aspergillus terreus, Arch. Biochem. 6 : 455-469, 1945. Lohrmann, W. : Untersuchungen liber die antagonistische Wirkung von Magnesium gegeniiber Bor und Quecksilber bei einigen Pilzen, Arch. Mikrohiol. 11 : 329-367, 1940. ESSENTIAL METALLIC ELEMENTS 85 McHargue, J. S., and R. K. Calfee: Effect of manganese, copper and zinc on growth and metabolism of Aspergillus flavus and Rhizopus nigricans, Botan. Gaz. 91: 183-193, 1931. McHargue, J. S., and R. K. Calfee: Effect of manganese, copper and zinc on the growth of yeast, Pkmt Physiol. 6: 559-5G6, 1931a. MacLeod, R. A., and E. E. Snell: Some mineral requirements of the lactic acid bacteria. Jour. Biol. Chem. 170: 351-3G5, 1947. Marsh, P. B. : Salts as antidotes to copper in its toxicity to the conidia of Sclerotinia fructicola, Phytopathology 35: 54-Gl, 1945. Molliard, M.: Influence d'une dose reduite de potassium sur les characteres physiologiques du Sterigmatacystis nigra, Compt. rend. acad. sci. 170: 949-951, 1920. Mulder, E. G. : On the use of microorganisms in measuring a deficiency of copper, magnesium and molybdenum in soils, Antonie van Leeuwenhoek 6: 99-109, 1939-1940. Mulder, E. G.: Importance of molybdenum in the nitrogen metabolism of micro- organisms and higher plants, Plant and Soil 1: 94-119, 1948. MuNTZ, J. A. : The role of potassium and ammonium ions in alcoholic fermentation, Jour. Biol. Chem. 171: 653-665, 1947. Nelson, J. M., and C. R. Dawson: Tyrosinase, Advances in Enzymol. 4: 99-152, 1944. NiCKERSON, W. J. : Inhibition of fungus respiration : a metabolic bio-assay method, Science 103 : 484-486, 1946. Perlman, D. : Some effects of metallic ions on the metabolism of Aerobacter aerogenes, Jour. Bad. 49: 167-175, 1945. Perlman, D.: On the nutrition of Sclerotium delphinii, Am. Jour. Botany 35: 360- 363, 1948. *Perlman, D.: Effects of minor elements on the physiology of fungi, Botan. Rev. 15: 195-220, 1949. Perlman, D., D. A. Kita, and W. Peterson: Production of citric acid from cane molasses. Arch. Biocheni. 11: 123-129, 1946. Rabinovitz-Sereni, D.: Influenza del magnesio suUo sviluppo di alcuni funghi, Dol. R. Staz. Veg. 13 : 203-226, 1933. Raulin, J.: Etudes chimiques sur la vegetation, Ann. sci. nat., Ser. V, 11: 93-299, 1869. Richards, O. W., and M. C. Tkoutman: Spectroscopic analysis of the mineral con- tent of yeast grown on synthetic and natural media. Jour. Bad. 3D : 739-746, 1940. Rickes, E. L., N. G. Brink, F. R. Koniuszy, T„ R. Wood, and K. Folkers: Crystalline vitamin B12, Science 107 : 396-397, 1948. RippEL, A., and G. Behr: Ueber die Verteilung des Magnesiums im Pilzmycel, Arch. Mikrohiol. 1 : 271-276, 1930. RippEL, A., and G. Behr: Ueber die Bedeutung des Kaliums im Stoffwechsel von Aspergillus niger, Arch. Mikrohiol. 5: 561-577, 1934. RoBBiNS, W. J., and A. Hervey: Response oi Pythiomorpha gonapodyoides to manga- nese. Bull. Torrey Botan. Club 71 : 258-266, 1944. Roberg, M.: Ueber die Wirkung von Eisen-, Zink-, und Kupfersalzen auf Aspergillen, Cent. Bakt., Abt. II. 74: 333-371. 1928. RoBERG, M.: Weitere Untersuchungen iiber die Bedeutung des Zinks fur Aspergillus niger. Cent. Bakt., Abt. II, 84: 196-230, 1931. Roberts, C. : The effect of iron and other factors on the production of pigment by the yeast Torulopsis pulcherrima, Atn. Jour. Botany 33 : 237-244, 1946, 86 PHYSIOLOGY OF THE FUNGI Shu, p., and M. J. Johnson: The interdependonce of modium oonstituents in citric acid production by submerged fermentation, Jour. Bad. 66: 577-585, 1948. Stark, W. H., P. J. Kolichov, and H. F. Willkie: Some factors affecting yeast propagation, Proc. Am. Soc. Brexving Chemists 4: 49-56, 1941. ■^Steinberg, R. A.: A study of some factors in the chemical stimulation of the growth of Aspergillus niger, Am. Jour. Botany 6: 330-372, 1919. Steinberg, R. A.: The nutritional requirements of the fungus Aspergillus niger, Bull. Torrey Botan Chib 62: 81-90, 1935. Steinberg, R. A.: Nutrient-solution purification for the removal of heavy metals in deficiency investigations with Aspergillus niger, Jour. Agr. Research 51 : 413-424, 1935o. Steinberg, R. A. : Relation of accessory growth substances to heavy metals, includ- ing molybdenum, in the nutrition of Aspergillus niger, Jour. Agr. Research 52: 439-448, 1936. Steinberg, R. A. : Role of molybdenum in the utilization of ammonium and nitrate nitrogen by Aspergillus niger, Jour. Agr. Research 55: 891-902, 1937. Steinberg, R. A.: The essentiality of gallium to growth and reproduction of Asper- gillus niger. Jour. Agr. Research 57: 569-574, 1938. Steinberg, R. A. : Correlations between biological essentiality and atomic structure of the chemical elements. Jour. Agr. Research 57: 851-858, 1938a. ♦Steinberg, R. A.: Growth of fungi in synthetic nutrient solutions, Botan. Rev. 5: 327-350, 1939. Steinberg, R. A.: Relation of carbon nutrition to trace element and accessory requirements of Aspergillus niger. Jour. Agr. Research 59: 749-763, 1939a. Steinberg, R. A.: A dibasal (minimal salt, maximum yield) solution for Aspergillus niger; acidity and magnesium optimum, Plant Physiol. 20 : 600-608, 1945. Steinberg, R. A. : Specificity of potassium and magnesium for the growth of Asper- gillus niger. Am. Jour. Botany 33: 210-214, 1946. ♦Steinberg, R. A.: Essentiality of calcium in the nutrition of fungi. Science 107: 423, 1948. Steinberg, R. A. : Growth on synthetic nutrient solutions of some fungi pathogenic to tobacco, Am. Jour. Botany 37: 711-714, 1950. Stout, P. R., and D. I. Arnon: Experimental methods for the study of the role of copper, manganese, and zinc in the nutrition of higher plants, Am. Jour. Botany 26: 144-149, 1939. Sumner, J. B., and G. F. Somers: Chemistry and Methods of Enzymes, Academic Press, Inc., New York, 1947. Thatcher, R. W.: A proposed classification of the chemical elements with respect to their function in plant nutrition, Science 79: 463-466, 1934. Waksman, S. a., and J. W. Foster: Respiration and lactic acid production by a fungus of the genus Rhizopus, Jour. Agr. Research 57: 873-900, 1938. Waring, W. S., and C. H. Werkman: Growth of bacteria in an iron-free medium. Arch. Biochem. 1 : 303-310, 1943. YoE, J. H., and L. A. Sarver: Organic Analytical Reagents, John Wiley «fe Sons, Inc., New York, 1941. YosHiMURA, F. : The action of some heavy metals upon the production of catalase in Aspergillus, Japan. Jour. Botany 10: 75, 1939-1940. Young, H. C, and C. W. Bennett: Growth of some parasitic fungi in synthetic culture media, Am. Jour. Botany 9: 459-469, 1922. CHAPTER 6 THE ESSENTIAL NONMETALLIC ELEMENTS OTHER THAN CARBON Fungus mycelium and spores are composed mainly of compounds of the nonmetallic elements. As a rule, more than 95 per cent of the fungus consists of hydrogen, oxygen, carbon, nitrogen, sulfur, and phosphorus. The nonmetallic essential elements are both structural and functional. The cell wall, which is composed mainly of chitin or cellulose, appears to be the most stable structure of the fungus. Protoplasm is highly labile, and the constituent compounds of protoplasm are continually undergoing destruction, repair, and synthesis. The various structural and functional compounds of organisms are in a state of continual flux (Hevesy, 1947). The turnover of essential elements in functional compounds is more rapid than in structural compounds. The terms utilization, assimilation, and dissimilation are frequently used in physiology. Utilization is a broad term and implies that an organism uses or gains some benefit from a specific substance. Fungi utilize water as a solvent but derive neither energy nor substance from it. Assimilation is the incorporation of substances or their degradation products into cellular materials. Assimilation implies synthesis. Dis- similation is the degradation, or breakdown, of complex compounds into simpler ones. This term is particularly applied to those processes such as alcoholic fermentation where intermediate metabolic products accumu- late in the medium. Frequently dissimilation must precede assimilation and may be considered as the first phase of utilization. HYDROGEN Hydrogen enters into the composition of nearly all organic compounds of interest to physiology except carbon dioxide. This is true of the organic nutrients used by fungi as well as of the fungus protoplasm and other cellular compounds. Elemental hydrogen is not used by fungi. All the hydrogen utilized by fungi is in chemical combination. Certain bacteria (hydrogen bacteria), however, are able to obtain energy by oxidizing hydrogen. The importance of water for all living organisms is so great that it seems impossible to conceive of life without water. The formula H2O is really the formula of steam. In the liquid state these simple molecules 87 88 PHYSIOLOGY OF THE FUNGI associate to form polymers. At room temperature water consists mostly of (H20)3, which is sometimes called trihydroL For a further discussion of water see Barnes (1937). The chemistry of life processes is largely confined to reactions which take place in the presence of water or in solution. In addition to being a solvent of remarkable powers, water is associated with the colloids which comprise protoplasm. Gortner (1949) has distinguished between "free" and "bound" water. Free water is mobile within the cell and serves as a solvent and for the purpose of translocation of the various products of metabolism. Bound water is firmly adsorbed by protoplasm, and in this form water does not freeze. This property of bound water enables cells to withstand low temperatures. The ability of fungus spores to with- stand low temperatures may well be due to their having most of their water content in the bound form. Water ionizes to form hydrogen (H+) and hydroxyl (0H~) ions. The effects of these ions on biological processes are so important that they will be discussed in detail in Chap. 8. OXYGEN Apparently none of the fungi are obligate anaerobes. Many are strictly aerobic, and some are facultatively anaerobic. An aerobic organism requires uncombined oxygen, while a facultative anaerobe may use combined oxygen in addition to free oxygen. The amount of oxygen required for optimum growth varies with the species. It is common to express the amount of oxygen available in terms of millimeters of mercury. Approximately 21 per cent of air is oxygen. The amount of oxygen may be regulated by controlling the air pressure within the culture vessel. If the barometric pressure is 740 mm. Hg, the partial pressure due to oxygen is ^Hoo X 740, or 155.4 mm. Hg. If the pressure within a culture vessel is reduced to 100 mm. Hg, the partial pressure of oxygen amounts to 21 mm. Hg. Tamiya (1942) has reported that Aspergillus onjzae has a maximum rate of respiration when the partial pressure of oxygen is 500 to 630 mm. Hg. Such partial pressures of oxygen are readily obtained by using oxygen-nitrogen mixtures. Ternetz (1900) reported the following effects of reduced oxygen supply on Ascophanus carneus: at 10 mm. Hg the mycelium grew with difficulty; at 20 mm. Hg growth was good, but no spores formed; at 40 mm. Hg some fructification occurred; at 120 to 140 mm. Hg growth was somewhat better than at atmospheric pressure. The ability of certain soil fungi to exist under conditions of low oxygen supply is important for survival. The amount of oxygen in soil depends upon the soil type and the amount of water present. Soil saturated with water contains but a trace of free oxygen. Hollis (1948) found Fusarium oxysporum to survive under essentially anaerobic conditions for 13 weeks, ESSENTIAL NONMETALLIC ELEMENTS 89 while F. eumartii perished within 3 weeks when exposed to the same condi- tions, The mycehum of F. oxysporuni grown under reduced oxygen ten- sion was abnormal in its morphology. For further information on the effect of reduced oxygen tension, see Fellows (1928) and Scheffer and Livingston (1937). Enormous amounts of sterile air must be supplied to the 10,000- to 15,000-gal. tanks used in the production of penicillin and other antibiotics. In the laboratory, aeration is provided by shaking machines of the rotat- ing or reciprocal type. Aeration under these conditions is more uniform than is possible in stationary cultures, W'here submerged and aerial hyphae obtain different amounts of oxygen. This was sho\Mi by Tamiya (1942) who reported that the enzyme systems of submerged mycelium of Aspergillus oryzae are more easily poisoned by cyanide than are those of aerial mycelium. In a broad sense, respiration denotes all the enzymatic processes which occur in cells involving a release of energy. There are two general ways in which energy is released by living cells: (1) Cells obtain energy from chemical reactions in which free oxygen is a reactant. The oxidation of metabolite molecules by this process is generally called respiration, or more specifically aerobic respiration. This process is characterized by the intake of free oxygen and the formation of carbon dioxide. If the com- pound being oxidized is composed of carbon, hydrogen, and oxygen only, the products are carbon dioxide, water, and energJ^ (2) Cells also obtain energy from chemical reactions in which free oxygen is not a reactant. This process is called anaerobic respiration, or fermentation. Metabolic processes of this kind are characterized by the production of carbon dioxide, the incomplete oxidation of substrate molecules, and the release of a small amount of energy. The reactions involved in the aerobic respiration of glucose may be summarized in a single equation: CsHisOe + 60.-^ 6CO2 + 6H2O + 673,000 cal. This equation gives no indication of the intermediate stages in this reac- tion or how the energy is utilized by the organism performing the oxida- tion. The number and variety of intermediate reactions do not affect the total amount of energy released. The reactions involved in the alcoholic fermentation of glucose are summarized in the following equation : CeHisOs-* 2CH3CH2OH + 2CO2 + 25,000 cal. This equation, like the preceding one, gives no indication of the inter- mediate reactions involved. To obtain the same amount of energy, more of a compound must be fermented than when it is completely oxidized. Not all of the energy released by either of these processes is available to the organism (Chap. 4). 90 PHYSIOLOGY OF THE FUNGI A knowledge of the amounts of oxygen consumed and carbon dioxide evolved by organisms is the basis of a useful method of study in many phases of physiology. The principles of such measurements are simple. In aerobic respiration both the oxygen and carbon dioxide may be meas- ured. The ratio of the moles, or volumes, of carbon dioxide evolved and oxygen used is called the respiratory quotient (R.Q.) and is written CO2/O2. From the respiratory quotient the nature of the substrate being oxidized may be deduced. A respiratory quotient of 1 is character- istic of aerobic oxidation of carbohydrate. The complete oxidation of a fat may be represented as follows: (C,8H3602)3C3H5 + 81.50-2^ 57CO0 + 55H.2O The respiratory quotient for this fat is 57/81.5, or 0.7. If fungus cells are suspended in a buffer in the absence of nutrients, and the respiratory quotient determined, it is possible to deduce the type of compound within the cells being used as a source of energy. Oxidation of the stored com- pounds within the cell is called endogenous respiration. The oxidation of substrate molecules from the medium is called exogenous respiration. Since both types of respiration may occur simultanously in the presence of nutrients, it is necessary, in order to determine exogenous respiration, to subtract the value for endogenous respiration from that obtained in the presence of nutrients. The rate and amount of respiration are determined by instruments known as respirometers. Various types of respirometers have been used to investigate different phases of fungus metabolism and nutrition. In principle a respirometer is a closed vessel of known volume in which fungus cells are suspended in a buffer or other solution. The carbon dioxide evolved is absorbed in a concentrated solution of potassium hydroxide. The change in volume due to the consumption of oxygen is measured by the use of suitable manometers. At the end of the experi- ment the amount of carbon dioxide evolved is measured after the potas- sium hydroxide solution is treated with a mineral acid. Carbon dioxide alone may be measured by passing a stream of carbon dioxide-free air through a culture and absorbing the carbon dioxide evolved in barium hydroxide or other suitable reagent. The results of such experiments are reported on the basis of the volumes of oxygen used and carbon dioxide evolved per milligram of dry weight per hour. These values are reported as Q02 and Qco2 (see Umbreit et at., 1945). A modern respirometer is illustrated in Fig. 16. The various manipula- tive details will not be discussed. For an adequate treatment of these see Umbreit etal. (1945) and Dixon (1943). These methods are extremely useful in studying a wide range of problems. Hawker (1944) used manometric techniques in studying the effect of excess thiamine on ESSENTIAL NONMETALLIC ELEMENTS 91 glucose utilization by Melanospora destruens and Phycomyces nitens. The papers of Siu and Mandels (1950) and Mandels and Siu (1950) should be consulted for details concerning a simple differential manometer. This manometer is designed to measure the respiration of intact growing cultures of filamentous fungi. Dorrell (1948) investigated the effect of Fig. 16. A constant-temperature bath and shaking device for micro respiration studies. (Courtesy of American Instrument Company.) dinitrophenol on endogenous and exogenous respiration of Fusarium graminearum (Gibberella zeae). As usually carried out, respiration experi- ments last only a few hours. The initial state of the cells or mycelium Table 16. The Effect of Age of Zygosaccharomyces acidifaciens Cells on the Amount of Aerobic Respiration (Nickerson and Carroll, Jour. Cellular Cornp. Physiol. 22, 1943. Published by permission of the Wistar Institute of Anatomy and Biology.) Age of cells, hr. Qo.* Glucose substrate No substrate (endogenous) 24 48 72 60 35 35.5 16 7.3 7.0 * Q02 equals lil O2 per hr. per mg. dry cells. 92 PHYSIOLOGY OF THE FUNGI used has a great effect on the results obtained. Nickerson and Carroll (1943) have indicated that the culture history of the cells used influences the amount of aerobic respiration. Some of their data for Zygosaccharo- myces acidifaciens are shown in Table 16. SULFUR Not all compounds which contain an essential element are equally useful. In fact, some compounds are useless because the essential ele- ment is unavailable. Among the factors which may affect availability is the state of oxidation of the essential element. This is particularly true of sulfur, phosphorus, and nitrogen. Among the organic compounds, structure is enormously important. The situation is further complicated in that not all fungi utilize the same compounds. Many examples of thi, will be cited in connection with nitrogen and carbon nutrition. Atten- tion must be given the sources of the essential elements as well as the uses fungi make of them. Sources of sulfur. This element is present in many types of com- pounds, both inorganic and organic. The state of oxidation of sulfur, as well as the specific structure of organic sulfur compounds, affects utiliza- tion. Sulfate sulfur, SO4"", is the most common source of sulfur used in media. Some fungi, however, require specific organic sources of sulfur. Steinberg (1936, 1941) has made an exhaustive study of sulfur sources for Aspergillus niger and reached the general conclusions that inorganic sulfur compounds containing oxidized sulfur are utilized, while sulfide and disulfide sulfur are not utiHzed. Of the organic compounds containing sulfur, the alkyl thioalcohols, sulfides, and disulfides are not used. Alkyl sulfonates and sulfinates are excellent sources of sulfur. Steinberg is of the opinion that oxidized sulfur is reduced to suKoxylate before it enters the normal metabolic channels. An exception to the nonutiliza- tion of reduced sulfur was noted for compounds which occur as normal metabolites, such as cysteine, cystine, methionine, and homocystine. These are assumed to enter normal metabolic channels without pre- liminary modification. An exception to this statement was noted with thiamine (thiazole sulfur), but the enormous (physiologically) amounts used may have upset the metabolic activities of the fungus. In spite of the general utility of sulfate sulfur in fungus nutrition, many fungi either utilize organic sulfur contained in natural metabolites to bet- ter advantage or require these compounds as a source of sulfur. Leonian and Lilly (1938) reported that the addition of cystine to a synthetic medium was necessary for the grovv^th of Saprolegnia mixta, Achlya con- spicua, Isoachlya monilijera, and Aphanomyces camptostylus. Since other naturally occurring sulfur-containing amino acids were not tested, it should not be concluded that these species are deficient for cystine. ESSENTIAL NONMETALLIC ELEMENTS 93 Volkoiisky (1933, 1934) observed that certain of the aquatic Phycocomy- cetes failed to utiHze sulfate sulfur. These species were Saprolegnia parasitica, Isoachlya monilifera, Achlya prolifera, A. polya7idra, A. oblon- gata, A. conspicua, Dichtyuchus monosporus, and Aphanomyces sp. A total of 26 isolates failed to utilize sulfate sulfur. This investigator (1933a) designates ability to utilize 6-valent sulfur as euthiotrophy and inability to utilize sulfate sulfur and ability to utilize reduced sulfur as parathiotrophy . Fries (1946) was able to induce mutation in Ophiostoma (Ceratostomella) rmiltianmilatum by irradiating the ascospores with X rays. Among these mutants 13 strains were unable to utilize sulfate sulfur. Only five of these strains regained this ability when cultivated on media containing sulfate. These parathiotrophic strains of 0. multiannulaium utilized ammonium sulfide as well as cystine and cysteine as sources of sulfur. From the fact that these mutants could utilize sulfide sulfur, it is evident that these strains were not deficient for specific sulfur-containing amino acids. Bonner (1946) has, however, found induced mutants of Peni- cillium to be deficient for specific sulfur-containing amino acids. Blasto- cladia pringsheimii has been reported to require methionine (Cantino, 1949). Fries (1948) has reported the occurrence of natural mutants of Ophi- ostoma multiannulatiim which require reduced sulfur, and also mutants which are unable to synthesize methionine. Of a total of 51,037 single- conidium cultures, 2 required reduced sulfur and 30 required methionine. The role of sulfur. The use fungi make of sulfur may be deduced from the sulfur-containing compounds which are known to occur in mycelium and spores. Among these are the proteins. In Chap. 4 it was noted that the activity of many enzymes depends upon the sulfhydryl or thiol group, ■ — SH. On hydrolysis, fungus protein yields the following sulfur-contain- ing amino acids: cystine, cysteine, and methionine. Sulfur is thus a structural element. Another sulfur-containing compound is the tripep- tide, glutathione, which is abundant in yeast. The formula for gluta- thione is given below: COOH CH2SH H2N— CH— CH2— CH2— CONH— CH— CONH— CH2— COOH This compound is sometimes represented by the symbol GSH. In spite of intensive investigation the role of this compound is not fully under- stood. Perhaps one of its functions is to protect sulfhydryl enzymes from inactivation. The probable mechanism of the biosynthesis of cystine has been studied using mutants of Aspergillus nidulans (Hockenhull, 1949). All these cystine-deficient mutants were able to utilize thiosulfate sulfur, methio- 94 PHYSIOLOGY OF THE FUNGI nine, and cystine. It was postulated that sulfate sulfur was first reduced to sulfite and then to sulfoxylate, which was assumed to dimerize to thiosulfate. The next reaction was believed to be between serine and thiosulfate to form cysteine S-sulfonate, which is then converted to cysteine. Cysteine on being oxidized forms cystine. Two vitamins, thiamine and biotin, contain sulfur. The role of these compounds will be considered in Chap. 9. In addition to the sulfur-con- taining amino acids and vitamins there is evidence that other types of organic sulfur compounds are formed by fungi. Raistrick and Vincent (1948) found that many strains and species of Aspergillus and Penicillium converted essentially all of the sulfate sulfur into organic sulfur com- pounds, but not all of these compounds were found in the fungus proteins. Penicillium chrysogenum excretes into the medium various unidentified organic sulfur compounds (Plumlee and Pollard, 1949). The function of these compounds is unknown. The reactions whereby a fungus transforms a single source of sulfur into these various compounds are obscure. When sulfate or other sources containing oxidized sulfur are utilized, it is necessary for the fungus to reduce the sulfur to its lowest valence. Schizophyllum commune has been shown to reduce sulfate to methyl mercaptan, CH3SH (Birkinshaw et al., 1942). This substance contributes to the characteristic odor of this fungus. PHOSPHORUS Raulin (1869) found phosphorus to be an essential element for Aspergil- lus niger. Omission of phosphate from his synthetic medium reduced the yield approximately 50 per cent. Phosphorus is essential for all forms of life. Phosphorus may be classified as a structural element in the sense that definite compounds containing this element have been isolated from fungi. Phosphorus compounds play an important role in the functions of chemical transformations and energy transfer. Sources of phosphorus. Apparently phosphorus is utilized only when it is in the form of phosphate. This element is taken up as phosphate and functions in this form, mainly in the form of phosphate esters. It will be recalled that there are several different phosphates. The formulas for the potassium salts are K3PO4, potassium orthophosphate ; KPO3, potassium metaphosphate ; and K4P2O7, potassium pyrophosphate. More complex phosphates than pyrophosphate occur. Orthophosphoric acid may be neutralized in three steps to produce the following types of salts: KH2PO4, monopotassium orthophosphate; K2HPO4, dipotassium orthophosphate; and K3PO4, tripotassium orthophosphate. All these salts furnish utiliz- able phosphate, but the effects of these three salts on the acidity of the medium are quite different. In addition to inorganic phosphates, the ESSENTIAL NONMETALLIC ELEMENTS 95 organic phosphates (esters) may also be used as sources of this element. Dox (1911-1912) investigated the assimilation of various phosphorus compounds by Aspergillus niger with the following results: Ortho-, meta-, and pyrophosphates supported excellent growth, as did such organic compounds of phosphorus as phytin, sodium glycerophosphate, sodium nucleinate, casein, and ovovitellin. Sodium hypophosphite (NaH2P02-H20) and sodium phosphite (Na2HP03-5H20) were not utilized and appeared to be toxic. Smith (1949) studied the phosphorus metabolism of MeruUus lacrymans and Marasmius chordalis in connection with the utilization of different carbon sources. In glucose medium M. lacrymans grew better when supplied w^ith inorganic phosphate, while M. chordalis grew miore rapidly when supplied with organic phosphorus (adenylic acid). On cellobiose medium M. lacrymans grew faster when supplied with organic phosphorus. The role of phosphorus. An idea of the manifold ways in which phos- phorus enters into fungus metabolism may be gained from the studies of Mann (1944, 1944a). Aspergillus niger was grown on a glucose-nitrate medium containing varying amounts of dipotassium orthophosphate. Some of Mann's data on the effect of two concentrations of phosphate are given in Table 17. Table 17. The Effect of Two Concentrations of Orthophosphate upon the Appearance, Sporulation, and Other Metabolic Functions of Aspergillus niger (Mann, Biochem. Jour. 38, 1944. Published by permission of the Cambridge University Press.) Characteristics of Grown in presence of Grown in presence of 5-day-old cultures 0.02% K2HPO4 0.2% K2HPO4 MyceUum Thin, white, smooth. Thick, yellowish. No Conidiophores present conidiophores Dry weight, mg. 460 1,092 Q02 of intact mycellium, lA 6.12 11.4 Total N, xng. 8.1 23.7 Total P, mg. 1.5 12.1 Thiamine, /xg 3.2 19.0 Riboflavin, ng 16.1 78.7 Nicotinic acid, ng 19.4 302.0 Medium Colorless YeUow From Table 17 it may be seen that suboptimal amounts of phosphorus affect the metabolism of A. niger in many ways besides diminishing growth. Nitrogen utilization was affected, and the synthesis of three vitamins (thiamine, riboflavin, and nicotinic acid) was greatly decreased. The ability of phosphorus-starved mycelium to utilize oxygen was dimin- ished, as shown by the lower Qo,- Mann also showed that utilization of 96 PHYSIOLOGY OF THE FUNGI phosphate by A. niger takes place only in the presence of oxygen. The utilization of phosphorus by yeasts, and presumably by other fungi which are capable of anaerobic respiration, may take place in the absence of oxygen. Various respiratory inhibitors such as iodoacetate, azide, and cyanide inhibited both respiration and phosphorus metabolism. This points to an intimate connection between carbohydrate and phosphorus metabolism. By analysis, ortho-, meta-, and pyrophosphates were found in the mycelium. Since only orthophosphate was supplied in the medium, it is shown that A. niger is capable of these transformations. Phosphorus appears to participate in almost every step in the anaerobic dissimilation of glucose into alcohol by yeast. Some of these steps may be common to other fungi. It is remarkable that the formation of alcohol by yeast and lactic acid in muscle should follow almost the same pathways. Phosphorus is required in the enzymatic transformation of glucose into alcohol and carbon dioxide (Harden, 1932). Sumner and Somers (1947) and Tauber (1949) have summarized the enzymatic reactions involved. Either starch or glycogen may be transformed into glucose-1-phosphate by enzymatic esterification. The shift of the phosphate radical to the other end of the glucose molecules leads to glucose-6-phosphate, which may also be formed by direct esterification of glucose. Glucose-6-phos- phate is transformed into fructose-6-phosphate and then into fructose- 1,6-diphosphate. Scission of a molecule of fructose-l,6-diphosphate yields dihydroxyacetone-1-phosphate and D-1-phosphoglyceric aldehyde. An equally long series of transformations leads to pyruvic acid, CHs — CO — COOH, Avhich on decarboxylation by the enzyme carboxylase yields acetaldehyde, which is enzymatically reduced by DPN-H2 to ethyl alcohol. Cocarboxylase and diphosphopyridine nucleotide (DPN) are coenzymes, both of which contain phosphorus. Gould et al. (1942) studied the formation of alcohol by Fusarnim tricothecioides and found the limited production of alcohol by this species was due to insufficient synthesis of diphosphopyridine nucleotide. Alco- hol production was increased 20- to 25-fold by the addition of either yeast extract or DPN to the medium. The paper of Semeniuk (1943-1944), which deals with the relation of phosphorus to glucose dissimilation by Chaetomium funicola, has an extensive bibliography (117 references). Nord and Mull (1945) have summarized a long series of papers on the physiology and biochemistry of Fusarium lini and reached the conclusion that fermentation by this fungus follows a pathway which does not involve the sugar phosphates. The review of Barron (1943) on the mechanisms of carbohydrate metabolisms contains much information about the role of phosphorus (219 references) in carbohydrate metabo- lism. The role of phosphorus compounds in the transfer of energy was noted in Chap. 4. ESSENTIAL NONMETALLIC ELEMENTS 97 Phosphorus enters into the composition of the nucleoproteins, which are found in the nucleus and cytoplasm of every cell. The nucleoproteins are conjugated proteins which consist of a protein moiety in combination with purine or pyrimidine nucleotides (nucleic acids). These nucleotides are important functional compounds and may be classified according to their heterocyclic components. The preliminary hydrolysis of purine and pyrimidine nucleotides involves the removal of phosphoric acid and the formation of nucleosides. Nucleosides on hydrolysis yield sugars, purines (adenine, guanine) or pyrimidines (cytosine, thymine, uracil). The nucleotides are also classi- fied according to the sugar moiety, i.e., D-ribose or D-desoxyribose. The nucleoproteins which contain D-ribose are mainly found in the cytoplasm, while D-desoxyribose characterizes the nucleoproteins of the nucleus. The Feulgen stain is used by cytologists to detect the presence of D-desoxyribose nucleic acid. Viruses, chromosomes, and genes consist largely of nucleoproteins. For a review of the role of nucleoproteins see Mirsky (1943). NITROGEN This essential element is used by fungi for functional as well as struc- tural purposes. The cell wall of many species, with the exception of the Oomycetes and yeasts, appears to be composed of chitin (Brian, 1949). Chitin is a linear polymer, similar to cellulose, of D-glucosamine. The amino group of glucosamine in chitin is acetylated. This substance makes up the exoskeleton of insects and Crustacea. It is interesting that the chitin formed by fungi, insects, and Crustacea appears to be the same substance. Protein, the basis of protoplasm, is composed of nitrogenous substances. Purines, pyrimidines, and some of the vitamins are also nitrogen-containing compounds. Not all nitrogen sources are equally suitable for all fungi. Fungi may be specific in the nitrogen sources they utilize. Our information on this subject, while extensive, is far from complete. The reports in the litera- ture which indicate that specific fungi are able to grow on a given source of nitrogen may be accepted with confidence, but the reported negative results are to be viewed with caution. Failure of a fungus to grow upon a given nitrogen source may mean only that the medium used did not con- tain the necessary growth factors, as in the case of Ophioholus graminis (See Chap. 2). Classification according to nitrogen sources used. Robbins (1937), Steinberg (1939, 1950), and others have classified the fungi according to their ability to utilize different sources of nitrogen. In the main Rob- bins's classification is as follows: (1) fungi able to utihze atmospheric nitrogen, nitrate nitrogen, ammonmm nitrogen, and organic nitrogen; (2) fungi able to utilize nitrate nitrogen, ammonium nitrogen, and organic 98 PHYSIOLOGY OF THE FUNGI nitrogen but not able to utilize atmospheric nitrogen; (3) fungi able to utilize ammonium and organic nitrogen but unable to utilize atmospheric or nitrate nitrogen; (4) fungi which are able to utilize only organic nitrogen and unable to utilize atmospheric, nitrate, or ammonium nitro- gen. Robbins recognized that the experimental conditions might affect the classification of some fungi. In spite of admitted imperfections the above classification is very useful in preparing media and in discovering the causes of failure of some fungi to grow on certain media. Nitrogen -fixing fungi. It has been shown to the satisfaction of all competent investigators that various genera of bacteria {Rhizohium, Azotobacter, Clostridium) contain species which are able to fix nitrogen. Table 18. Nitrogen Fixation by Phoma betae and Azotobacter vinlandii (Duggar and Davis, Ann. Missouri Botan Garden 3, 1916.) Inoculated flasks Uninoculated flasks Mg. N fixed per flask Organism IVIg. N per flask Ave, Mg. N per flask Ave. Aspergillus niger (30 days) Phoma betae (89 days) Azotobacter vinlandii (28 days) 62.510 62.545 63.140 31.010 31.360 46.515 46.480 46.445 62 . 732 31.185 46 . 480 62.510 62.335 62.300 25.585 25.655 5.810 6.405 62.382 25.620 6.108 0.350 5.565 40.372 No such agreement exists regarding fungi. Much of the early work on nitrogen fixation by fungi was done without using proper precautions. However, in several instances the experimental methods appear to be beyond reproach. Duggar and Davis (1916) cultured Phoma betae and Aspergillus niger in Kjeldahl flasks and determined the nitrogen content after growth without removing either the mycelium or medium prior to digestion. Two types of controls were used. A number of uninoculated flasks which had been stored under the same conditions as the inoculated flasks were analyzed for nitrogen at the end of the experiment. A culture of Azotobacter vinlandii served as a positive control. The data in Table 18 show that A. niger did not fix nitrogen, while P. betae and A. vinlandii did. However, the nitrogen-fixing power of P. betae was slight compared with that of A. vinlandii. In addition, the following fungi were tested for ability to fix nitrogen, with negative results: Macrosporium commune, Penicillium digitatum, P. expansum, and Glomerella gossypii. For further ESSENTIAL NONMETALLIC ELEMENTS 99 references to nitrogen fixation by filamentous fungi see Wolf and Wolf (1947) and Buchanan and Fulmer (1930). So far as we are aware, only one study of nitrogen fixation by fungi using modern isotopic techniques (Tove et at., 1949) has been published. Phoma causarina was grown on a sucrose-salts medium in oxygen and nitrogen enriched with N^^. Growth was slow and sparse under these conditions, but some N^^ was fixed. These authors state that the isotopic method is about 100 times more sensitive than the Kjeldahl procedure used by other investigators. While it is probable that only a relatively few fungi are able to fix nitro- gen, the importance of biological nitrogen fixation is so great that further investigations with modern techniques are desirable. Long ago Ternetz (1907) reported that five species of Phoma isolated from roots of Ericaceae fixed significant amounts of nitrogen. For a discussion of nitrogen fixa- tion by bacteria see Wilson (1940). Fungi utilizing nitrate nitrogen. Nitrates occur in the soil and thus are a ''natural" source of nitrogen. A fungus which utilizes nitrate nitrogen (NOs") must be able to reduce the nitrogen to the oxidation level of ammonia. We may assume that failure of a fungus to utilize nitrate nitrogen is coupled with inability to perform this reduction. According to Robbins (1937) no instances have been recorded in the literature of an organism being able to utilize nitrate nitrogen and unable to utihze ammonium nitrogen. This does not mean that fungi which are able to utihze nitrate nitrogen will grow at the same rate on ammonium nitrogen, or that all sources of organic nitrogen will be as favorable as nitrate nitro- gen. Yeasts as a rule do not utilize nitrate nitrogen. The following is a partial list of fungi which have been reported or observed to utilize nitrate nitrogen: Armillaria rnellea C. velutipes Ascobolus denudata Cordyceps militaris A. leveillei Dendrophoma obscurans Ascochyta pisi Dothidella quercus Aspergillus spp. Fusarium spp. Botryotinia convoluta Glomerella cingulata Botrytis allii Gyrnnoascus setosus B. cinerea Helminthosporium spp. Cephalothecium roseum Lambertella corni-maris Cercospora apii Lentinus tigrinus C. beticola Macrosporium sarcinaeforme Chaetomiiim cochlioides Marasmius Julvobidbillosus C. convolutum Neocosmopara vasinfecta C. globosum Ophiobolus graminis Colletotrichum lagenarium 0. miyabeanus C. lindeniuthianum Penidllium spp. Collybia tuberosa Phoma apiicola 100 PHYSIOLOGY OF THE FUNGI P. betae S. sclerotioruni Pleurage curvicolla Sderotium bataticola Pyronema confluens Septoria nodorum Pythiomorpha gonapodyoides Sordaria fimicola Pythium debaryanum Sphaeroholus stellatus P. intermedium Sphaeropsis malorum P. irregular e Trichoderma lignorum Rhizodonia solani VerticiUium albo-atrum Sderotinia minor Xylaria mali Several of the species in the above Hst were reported by Young and Bennett (1922) and others by Robbins and Kavanagh (1942). Some reports are found in the papers of various authors, while some of the fungi have been observed in our laboratory (see Fig. 17 for illustrations). Fungi which utilize ammonium nitrogen. In the nitrogenous com- pounds found in fungi the nitrogen is in the same state of oxidation as in ammonium compounds. The following is a partial list of fungi which have been reported or observed to require ammonium or organic nitrogen and to be unable to assimilate nitrate nitrogen: Absidia coerulea M. putillus A. cylindrospora M. ramealis A. diibia M. rotula A. glauca M. scorodonius A. orchidis Monilinia frudicola Basidiobolus ranarum Mortierella rhizogena Ceratostomella fimbriata Mucor flavus C. ulmi M. hiemalis Choanephora cucurbitarum M. nodosus Cyathus striatiis M. pyriformis Endothia parasitica M. saturninus Lenzites trabea M. stolonifer Marasmius alliaceus M. stridus M. androsaceus Phycomyces blakesleeanus M. chordaiis Pleurotus ostreatus M. epiphyllus Rhizophlydis rosea M. foetidis Rhizopus nigricans M. graminum R. oryzae M. performis Sporodina grandis M. personatus Zygorrhynchus moelleri A number of fungi in the above list were listed by Robbins (1937). The studies on Marasmius are reported by Lindeberg (1944). Others are reported by various authors, while some have been observed in our laboratory. Obviously, this list is far from complete, and numerous common fungi have been omitted from both this and the previous list because of lack of definite information regarding their ability to utilize nitrate nitrogen. ESSENTIAL NONMETALLIC ELEMENTS 101 Fungi which utiUze only organic nitrogen. Certain fungi are unable to utilize nitrogen except in the form of amino acids, peptides, and mixtures of these compounds such as peptone. The use of organic nitrogen does not extend to all organic compounds which contain this element. Many A B C D Fig. 17. Growth of two fungi on four media differing in nitrogen source. .4, no nitrogen added; B, potassium nitrate; C, ammonium tartrate; D, asparagine. Above, Helminthosporium sativum; below, Ceratostomella fimbriata. of the early reports claiming utilization of organic nitrogen only by various species have been found to be in error. All the early work where peptone was the nitrogen source used is to be suspected because the need for growth factors was not recognized. The use of other complex nitrogen sources such as proteins makes interpreta- tion doubtful for the same reason. However, in the case of amino-acid- deficient fungi a portion of the nitrogen source must be supplied in the 102 PHYSIOLOGY OF THE FUNGI form of a particular amino acid. Cantino (1949) found that Blastocladia pringsheimii is deficient for methionine and perhaps other amino acids. Presumably other amino acids are used to supply a portion of the metabolic nitrogen of this species. The same situation may exist in the nitrogen utilization of amino-acid-deficient mutants of Neurospora. Leonian and Lilly (1938) reported Coprinus lagopus and Pleurotus corticatus to grow on a mixture of five amino acids and not on ammonium nitrate as a source of nitrogen. Inorganic sources of nitrogen. The nitrates commonly used in prepar- ing media are potassium nitrate, sodium nitrate, and calcium nitrate. These salts are equivalent in so far as they supply the same kind of nitro- gen. They are not equivalent in that different cations are involved. Calcium ion may precipitate a varying amount of phosphate, depending upon the concentrations of the two ions and the pH of the medium. Some fungi utilize nitrite (N02~) nitrogen. Blakeslea trispora makes some growth on nitrite nitrogen (Leonian and Lilly, 1938). Owing to the instability of nitrites in acid solution and the destructive effect of nitrous acid on proteins and amino acids, nitrite nitrogen is little used in making media. Nitrite is produced by many fungi from nitrate and may accumu- late in the medium under certain conditions. The toxic effect is related to the pH of the medium, being greatest at low pH. Wirth and Nord (1942) attributed the accumulation of pyruvic acid in the nitrate medium on which Fusarium lini grew to the presence of the nitrite, which inac- tivated thiamine pyrophosphate (cocarboxylase). Yeasts utilize nitrate nitrogen poorly as a general rule. Pirschle (1930) studied the relative value of nitrate and ammonium nitrogen for a yeast and concluded that poor utilization of nitrate nitrogen was due in part to the accumulation of nitrite in the medium. This was shown by the yields of aerated and nonaerated cultures on media containing nitrate and ammonium nitrogen as well as by analyses of the culture medium for nitrite. Aeration prevented the accumulation of toxic amounts of nitrite or its decomposition product nitrogen trioxide. In other experiments Pirschle showed that nitrite inhibited the growth of yeast on ammonium nitrogen. By adding sufficient nitrite to a medium containing ammonium sulfate, growth was depressed below that obtained on potassium nitrate. How far these conclusions may be applied to other fungi which do not utilize nitrate nitrogen is not known. Inorganic and organic ammonium salts are equivalent in that they furnish inorganic nitrogen; i.e., ammonium ion. The nitrogen of all ammonium salts is the same, but the physiological effects of the anions are not. The ammonium salts of strong inorganic acids generally tend to make a culture medium more strongly acidic than when an ammonium salt of a weak acid is used. However, the situation is far more compli- ESSENTIAL NONMETALLIC ELEMENTS 103 cated than this simple theory Yv'ould predict. It should be emphasized that nitrates and ammonium salts have opposite effects on the acidity of culture media. Other conditions being equal, as nitrate ions are con- sumed, the culture medium becomes more alkaline, while as ammonium ions are utilized, the culture medium becomes more acid. Before considering the ammonium salts of the organic acids, the use of ammonium nitrate should be mentioned. Both ions contain nitrogen, a feature which has led many investigators to use it in media. If a fungus is able to utilize both kinds of nitrogen, the pH of the medium will be somewhat stabilized. This salt should not be used if the purpose of an experiment is to determine whether a fungus can utilize either one or the other or both forms of nitrogen. Some fungi apparently use nitrate nitrogen in preference to ammonium nitrogen when both are supplied in the medium. Fusarium lini appears to be such a fungus (Wirth and Nord, 1942). Table 19. The Effect of Various Organic Acids on the Growth of Four Fungi on Media Containing Ammonium Nitrate Initial pH 5.5. Figures are milligrams of mycelium produced. (Leonian and LiUy, Am. Jour. Botany 27, 1940.) Organic acids, 0.02M Mucor raman- nianus Phythium ascophallon Pythiomorpha gonapodyoides Phycomyces blakesleeanus Control Acetic Lactic 77 144 154 142 135 158 174 152 8 19 34 117 23 156 56 0 8 40 113 157 76 180 112 0 27 144 121 Succinic Glutaric Fumaric 165 144 189 Tartaric Citric 149 171 M etarrhizium glutinosum {Myrothecium verrucaria) grew well on nitrate nitrogen alone and poorly on ammonium nitrogen (Brian et al., 1947). Ammonium nitrogen inhibited growth of this fungus, whether nitrate was present or not. Growth was equally poor on ammonium nitrate and ammonium sulfate. Since this fungus grew well on media containing nitrate as the sole source of nitrogen, these authors have questioned the common belief that all fungi which are able to utilize nitrate nitrogen can also utihze ammonium nitrogen. Most fungi appear to utihze ammonium nitrogen before nitrate nitrogen when both are supplied in the medium, but this is not universal. Rippel (1931) found the pH of the medium to determine which form of nitrogen was utilized by Aspergillus niger and A. oryzae. Additional examples are given by Foster (1949). The utilization of ammonium and some forms of organic nitrogen may 104 PHYSIOLOGY OF THE FUNGI be modified by the presence of other compounds in the medium. Among these the organic acids, especially the four-carbon dicarboxylic acids, play an important role. This subject has been studied by Leonian and Lilly (1940), Burkholder and McVeigh (1940), Brian et at. (1947), and Bernhard and Albrecht (1947). The data in Table 19 illustrate the effect of organic acids on the amount of growth of four fungi. Succinic and fumaric acids were most uniform in their effect on nitrogen assimilation. Figure 18 shows the reciprocal effect of varying amounts of 200 ~~^^ > c ^.-^ 1 1 150 V < / \ ^2.5g.Nh '4NO3/I 100 / \ 7. succinic ac id /I C 50 < \ °( ^ D 1. D g. NH4r 2 OO3/I 0 3.0 3.0 2.0 1.0 g. succinic acid/l Fig. 18. The reciprocal effect of varying amounts of succinic acid (ammonium nitrate constant) and ammonium nitrate (succinic acid constant) on the growth of Phycoviyces blakesleeanus. (Drawn from data of Leonian and Lilly, Am. Jour. Botany 27: 22, 1940.) succinic acid and ammonium nitrogen on the growth of Phycomyces blakesleeanus, which does not utilize nitrate nitrogen. The amount of growth, within certain limits, is directly proportional to the amount of succinic acid in the medium. Brian et al. (1947) have suggested on the basis of studies on Myro- thecium verrucaria that a definite antagonism exists between the metabolic pathways involved in nitrate and ammonium utilization, and in the presence of ammonium nitrogen the nitrate pathway is blocked. Ammo- nium nitrogen is poorly utilized unless certain organic acids are present in the medium. Malic acid has no effect on utilization of nitrate nitrogen. ESSENTIAL NONMETALLIC ELEMENTS 105 These authors suggest that different pathways of carbohydrate iitihzation may be followed, depending upon whether nitrate or ammonium nitrogen is present. Organic sources of nitrogen. Of the vast number of organic com- pounds which contain nitrogen the ones of interest in fungus nutrition are those which occur naturally. A few exceptions will be noted later. In practice, this means proteins and the products of protein hydrolysis. The following steps in protein hydrolysis have been recognized : protein -^ metaprotein — > proteoses — > peptones — > peptides — > amino acids. Pep- tone, which is a complex mixture of peptides and amino acids, is frequently used as a nitrogen source in media. According to Gortner (1929), peptones are neither coagulated by heat nor precipitated by saturating a solution with ammonium sulfate, properties which distinguish peptones from proteins, metaproteins, and proteoses. Since peptides having some 11 amino-acid residues are precipitated by ammonium sulfate, it may be deduced that the peptides in peptone have on the average 10 or less amino-acid residues. Peptone is a useful source of nitrogen when it is desired to culture a large number of species upon a single medium. A part of its virtue may be ascribed to its complex nature, for a mixture of nitrogen sources may be better utilized than a single source. Peptone also contains most of the water-soluble vitamins (Stokes et al., 1944). Most of the amino acids which have been isolated from proteins are listed in Table 20. In addition, the amides of aspartic and glutamic acids are included. These compounds are found free in many plants and are thus available to the fungi in nature. These amino acids are not of equal value in fungus nutrition. The relative value of 24 amino acids for 14 fungi was tested by Leonian and Lilly (1938) who found no one amino acid w^as best for all these species. Steinberg (1942) made an extensive study of growth of Aspergillus niger on 22 amino acids. Seven were excellent sources of nitrogen for A. niger: alanine, arginine, aspartic and glutamic acids, glycine, proline, and hydroxyproline. Steinberg expressed the opinion that the seven amino acids which supported the most growth of A. niger are those which are synthesized first (primary amino acids) by this fungus and from which the other amino acids (secondary amino acids) are normally formed. It is assumed that the "primary" amino acids enter directly into the metabolic pathways, while the "secondary" amino acids must undergo preliminary deamination before use. The primary amino acids are probably not the same for all fungi. Lilly and Leonian (1942) investigated the effect of nitrogen source on the growth of 10 strains of Saccharomyces cerevisiae. The data in Table 21 show clearly that different amino acids vary in effectiveness, and that different strains of the same organism respond differently to the same source of nitrogen. 106 PHYSIOLOGY OF THE FUNGI Table 20. Common Names and Formulas ok Some Alpha-amino Acius Isolated FROM Proteins and of Some Amides Found in Plants Monoamine dicarboxylic acids: Aspartic acid: HOOC— CH2— CHCNHo)— COOH Glutamic acid: HOOC— CHo— CH.— CH(NH2)— COOH Amides of monoamino dicarboxylic acids: Asparagine : Glutamine: Basic amino acids: Argiiiine*: Lysine*: Histidine * : NH2OC— CH2— CH(NH2)— COOH NH2OC— CH2— CH,— CH(NH2)— COOH NHo— C(=NH)— NH— CHo— CH2— CH2— CH(NH2)- NH2— CH2— CH2— CH2— CH2— CHCNH,)— COOH CH -COOH N ^ NH CH===C— CH2— CH(NH2)— COOH Monoamino monocarboxylic acids: Glycine: Alanine : Valine * : Leucine*: Isoleucine*: Phenylalanine * : Serine : Threonine*: Tryptophane '* Tryosine : CHoCNH.)— COOH CH3— CH(NH2)— COOH (CH 3) 2— CH— CH (NHo)— COOH (CH3)2— CH— CH2— CH(NH2)— COOH CH3— CH2— CH(CH3)— CHCNH.)— COOH CeHs- CHo— CH (NH2)— COOH CH2(0H)— CH(NH2)— COOH CH3— CH(OH)— CH(NH2)— COOH C— CH2— CH(NH2)— COOH CH NH HO^^^ CH2— CH \ CH2— CH(NH2)— COOH Proline: CH2 CH— COOH NH CHOH -CH2 Hydroxy proline: CHo CH— COOH NH Sulfur-containing amino acids: Cysteine: CHsCSH)— CHCNHo)- COOH Cystine: HOOC— CH(NH2)— CH2— S— S— CH2— CHCNHa)- COOH Methionine*: CH2(SCH3)CH2— CHCNH.)- COOH * The 10 amino acids reported by Rose (1938) as essential for the nutrition of the white rat. Physiological specificity extends to the configuration as well as the composition of the molecule. Optical isomers (enantiomorphs) usually have different physiological properties. A mixture of amino acids may or may not be utilized better than a single amino acid. The effect of one amino acid on the utilization of another varies with the amino acids ESSENTIAL XON METALLIC ELEMENTS 107 involved and the specific fungus used. Leonian and Lilly (1940) tested the growth of Phycomyces blakesleeanus upon five single amino acids and upon a mixture of these five amino acids with the following results : mix- ture of five amino acids, 214; asparagine, 209; DL-alanine, 151; arginine, 50; aspartic acid, 203; glycine, 201; and glutamic acid, 189 mg., respec- tively. Arginine is a poor nitrogen source for P. blakesleeanus, but the presence of arginine in the amino-acid mixture did not depress growth. More complex relations were found with yeast (Lilly and Leonian, 1942). Ten strains of yeast were grown upon media containing a mixture of six amino acids (aspartic and glutamic acids, arginine, asparagine, alanine, and leucine). Upon this mixture of amino acids two strains grew as well as or better than upon the best single amino acid (aspartic acid). The Table 21. Comparison of Various Soi'rces of Nitrogen for Six Strains of Yeast Milligrams of dry yeast cells produced in 72 hr. Each culture received 8 mg. of X. (Lilly and Leonian, Proc. West Va. Acad. Sci. 16, 1942.) Nitrogen source Ammonium sulfate Urea L- Aspartic acid . . . L-Aspargine Glycine DL-Norleucine Yeast strain 18.7 33.2 60.7 49.4 3.0 29.3 21.2 31.5 59.9 45.8 1.2 17.6 22.3 32.9 65.6 50.0 2.0 33.0 17.5 27.3 62.0 49.2 2.1 18.4 21.7 32.0 52.4 47.6 1.0 1.2 23.8 35.1 70.6 35.0 1.1 4.2 amount of growth of one strain was 70.6 mg. on aspartic acid alone, while on the amino-acid mixture only 38.6 mg. was produced. Omission of asparagine from the mixture increased the yield to 52.0 mg. These results show that the effects of multiple nitrogen sources upon growth, and perhaps other functions, are complex. Organic acids, especially the four-carbon dicarboxylic acids, affect the utilization of some amino acids much as they do that of ammonium com- pounds. Phycomyces hlakesleeamis on a medium containing arginine produced 43 mg. of mycelium per flask. Addition of 0.1 per cent succinic acid to the medium increased the yield to 192 mg. (Leonian and Lilly, 1940). Nitrogen utilization by the fungi has been studied for almost a century, but many of the problems involved are not yet solved. Brenner (1914) has reviewed the early work in this field, especially with reference to the divergent views of Raciborski and Czapek on the mode of utilization of amino acids. Raciborski held that amino acids were deaminat^d before 108 PHYSIOLOGY OF THE FUNGI utilization, while Cznpek believed that amino acids were utilized directly. Both processes arc doubtless involved, and only prolonged study of specific fungi and various nitrogen sources will permit elucidation of these questions. One of the main uses of nitrogen is in the synthesis of proteins. With the exception of certain amino acids (primary amino acids) and ammonia, most nitrogen sources undergo modification before entering the synthetic metabolic pathways. Nitrates, nitrites, and hydroxylamine are pre- sumably reduced to ammonia before assimilation. Those amino acids (secondary amino acids) which do not enter directly into the metabolic pathways leading to the synthesis of protein are probably deaminated. Burk and Horner (1939) have listed the types of deamination performed by fungi as follows: 1. Deamination by hydrolysis: H2O R— CH(XH.:)— COOH > R— CH(OH)— COOH + NH3 2. Deamination by hydrolysis followed by decarboxylation: H2O R— CHCNHo)— COOH > R— CH2OH + CO2 + NH3 3. Oxidative deamination: MO, R— CHCNHa)— COOH > R— CO— COOH + NH, The production of higher alcohols, "fusel oil," is due to hydrolytic deamination and decarboxylation of various amino acids, especially leucine, which yields isoamyl alcohol. Various species of filamentous fungi, especially those which produce alcohol, are capable of the same reactions. The following amino acids are converted by yeasts into alco- hols having one less carbon than the parent amino acid: leucine, isoleucine, phenylalanine, trytophane, and valine. Wirth and Nord (1942) indicate that Fusarium lini oxidatively transforms alanine into pyruvic acid. For further information on the process of deamination by yeast, see Thorn (1937). The process of deamination releases nitrogen in the form of ammonia, which is utilized by most fungi. It seems probable that the synthesis of amino acids is the next step in protein formation. The formation of primary amino acids may result from the reaction of ammonia with certain alpha-keto acids (pyruvic, oxalacetic, and ketoglutaric) ; this is essentially the reverse of oxidative deamination. This process may be fo.'mulated as follows: R— CO— COOH + NH3 -> R— C(=NH)— COOH + H. -^ R— CHCNH.)— COOH In addition, yeasts are able to add ammonia to fumaric acid to form aspartic acid (Haehn and Leopold, 1937). The role of the four-carbon dicarboxylic acids in nitrogen assimilation may be explained on the basis that these acids are transformed into kcto acids. Brian et al. (1947) have assumed that those fungi, such as Phycomyces hlakesleeanus and Myro- ESSENTIAL NONMETALUC ELEMENTS 109 thecium verrucaria, which make hmited growth on ammonium nitrogen do so because they are unable to synthesize in adecjuate amounts the neces- sary three-, four-, and five-carbon keto acids. The interrelation among various dicarboxylic acids is shown in schemes IV, VIII, and IX. The reactions discussed above account for the synthesis of only a few of the 20 or so amino acids found in fungus protein. Another type of reac- tion may account for the synthesis of secondary amino acids. This is called the transamination reaction and may be represented as follows : R— CO— COOH + R'— CH(NH2)C00H -^ R— CH(NH2)C00H+R'— CO— COOK According to Roine (1947), Torulopsis utilis has the necessary enzymatic mechanisms for the synthesis of the following amino acids by transamina- se 30 40 50 Time in minutes 60 70 Fig. 19. Amounts of soluble nitrogen compounds found in the trichloroacetic acid extract as a function of time. Data are based on 100 ml. of yeast suspension, or about 5 g. fresh yeast. Curve 1 represents total soluble nitrogen, curve 2 total amide nitrogen, curve 3 alanine nitrogen, and curve 4 dicarboxylic-amino-acid nitro- gen. (Courtesy of Roine, Ann. Acad. Sci. Fennicae 26: 63, 1947.) tion: aspartic acid, glutamic acid, alanine, valine, leucine, and isoleucine. For a general review of the transamination reaction, see Herbst (1944). Roine (1947) has obtained experimental evidence which indicates that in Torulopsis utilis the primary amino acids are formed first and that the secondary amino acids are then formed from them. This evidence w^as obtained by analyzing the nonprotein nitrogen fraction which was extracted from cells of various ages with trichloroacetic acid (a protein precipitant) . Nitrogen-starved cells of T. utilis were suspended in carbo- hydrate-free medium which contained ammonium nitrogen. The culture was aerated. Every 10 min. a portion of the crop was harvested, and the distribution of nitrogen compounds in the trichloroacetic acid extract was determined. Figure 19 show clearly that the first stages of protein syn- thesis consist in the formation of monoamino dicarboxylic acids, their no PHYSIOLOGY OF THE FUNGI amides, and alanine. It may be assumed that the amides of gkitamic and aspartic acid function in yeast as nitrogen carriers, as they do in green plants. Preformed amino acids are probably used in protein synthesis. In principle this process is the reverse of hydrolysis. Many complex chem- ical reactions are involved. Proteins vary in complexity, the simplest having molecular weights in the neighborhood of 16,000 to 17,000. The molecular weight of some proteins is said to be greater than 1,000,000, and tobacco mosaic virus protein is estimated to have a molecular weight of 40,000,000. In spite of these enormous molecular weights, a good deal is known about the structure of proteins. Fundamentally, a pro- tein consists of amino-acid residues joined together by peptide linkages, — CH2 — NH — CO — . Since different proteins have highly specific properties which depend upon the molecular structure, the synthesis of these compounds involves a systematic linking together of amino-acid residues in a definite pattern. For reviews of protein structure the reader is referred to Bull (1941) and Astbu^y (1943). The general pathways of nitrogen utilization by fungi are shown in scheme III. Scheme III. Possible Pathways of Protein Synthesis from Various Sources OF Nitrogen Nitrates — ■ > Ammonia > Primary amino acids Secondary amino acids Ammonia — i Secondary amino acids Primary amino acids- Peptides i Polypeptides i. Protems OTHER NONMETALLIC ELEMENTS It is not known whether fungi require nonmetallic elements other than hydrogen, oxygen, sulfur, phosphorus, and nitrogen. Boron and iodine are frequently added to culture media, but good evidence of their essen- tiality for fungi appears to be lacking. Sodium chloride is frequently added to media, but neither sodium nor chlorine, so far as is known, is essential for the fungi. In nature fungi come in contact with many nonessential elements. Some of these may be metabolized. Others may modify the life processes of the fungi by their toxic action or by other means. Chlorine is found in various compounds synthesized by fungi, e.g., non-ionic chlorine is found in chloramphenicol, one of the newer antibiotics. Many species of fungi metabolize arsenic. Penicillium hrevicaule, among other species, pro- ESSENTIAL HON METALLIC ELEMENTS 111 duces a volatile, toxic, organic arsenic compound, trimethylarsine, (CH3)3As, which has an odor resembling garlic. In the past P. hrevicaule has been recommended for the detection of arsenic compounds in forensic medicine. This microbiological test for the presence of arsenic is said to ))e many times as sensitive as the ]\Iarsh test. The early work on the utilization of arsenic compounds by fungi is reviewed by La Far (1911) and more critically by Challenger et al. (1933). P. hrevicaule also pro- duces dimethyl selenide from selenium compounds (Challenger and North, 1934). SUMMARY The classification of essential elements as structural or functional may be misleading in that an element usually plays many roles. This is especially true of the essential nonmetallic elements. With the exception of carbon dioxide all the organic compounds used by or contained in fungi contain hydrogen. One of the most important hydrogen-containing compounds is water. This compound is associated with proteins in the form of bound water, and it functions as a solvent in which most if not all biochemical reactions take place. Water enters into many reactions, particularly in the hydrolytic processes of ''diges- tion." Apparently fungi do not utilize free hydrogen. None of the fungi appear to be obligate anaerobes. Many are faculta- tive anaerobes, while some appear to be strict aerobes. Free oxygen is used by the fungi in respiration, chiefly as an acceptor of hydrogen. The facultative anaerobes have another mechanism of oxidation which does not involve free oxygen. This is called anaerobic respiration, or fermen- tation. The rate and amount of growth and sporulation and the meta- bolic by-products of a given fungus are affected by the oxygen supply. The problem of specificity arises in connection with the form of sulfur utilized. Most fungi utilize sulfate sulfur, but some require reduced sul- fur. Other species are unable to synthesize specific sulfur-containing amino acids, especially methionine. Sulfur enters into the composition of enzymes and other proteins, peptides, and at least two vitamins. The fungi utilize phosphorus in the form of phosphate salts and esters. Some specificity in the different sources of phosphate has been found Phosphate esters enter into a wide variety of enzymatic reactions, and many coenzymes are phosphate esters. It is thought that certain phosphate esters act to transfer chemical energy to certain enzymatic reactions. Phosphorus enters into the com- position of proteins, especially the nucleoproteins, which are found in the nucleus or cytoplasm of every cell. Viruses and genes are thought to consist largely of nucleoproteins. Fungi differ in ability to utilize different forms of nitrogen. A few utilize atmospheric nitrogen; many utilize nitrate nitrogen; and a still 112 PHYSIOLOGY OF THE FUNGI greater number utilize ammonium nitrogen. All species are abletoutilize some form of organic nitrogen. Other constituents in media, especially the four-carbon dicarboxylic acids, modify the availability of ammonium nitrogen and certain amino acids. Not all amino acids are of equal value in fungus nutrition. The primary amino acids are those which enter directly metabolic pathways, while secondary amino acids are deaminated before the nitrogen is used. Most of the nitrogen utilized by fungi enters into the synthesis of pro- teins. The primary amino acids are formed first, and the secondary amino acids are formed from primary amino acids. Proteins are the most complex compounds synthesized by living cells. Many of the vitamins and other essential metabolites also contain nitrogen. REFERENCES AsTBUKY, W. T. : X-rays and the stoichiometry of the proteins, Advances in Enzymol. 3: 63-108, 1943. Barnes, T. C: Textbook of General Physiology, The Blakiston Company, Phila- delphia, 1937. Barron, E. S. G. : Mechanisms of carbohydrate metabolism. An essay in compara- tive biochemistry, Advances in Enzymol. 3: 149-189, 1943. Bernhard, K., and H. Albrecht: Stoffwechselprodukte des Mikroorganismus Phycomyces Blakesleeanus in glucose haltiger Nahrlosung und Untersuchungen liber das Wachstum dieses Schimmelpilzes bei verschiedenen Stickstoffquellen, Helv. Chim. Acta 30 : 627-632, 1947. Birkinshaw, J. H., W. P. K. Findlay, and R. A. Webb: Biochemistry of wood-rot- ting fungi. 3. The production of methyl mercaptan by Schizophyllum com- mune Fr., Biochem. Jour. 36: 526-529, 1942. Bonner, D.: Production of biochemical mutants in PeniciUium, Am. Jour. Botany 33:788-791, 1946. Brenner, W.: Die Stickstoffnahrung der Schimmelpilze, Cent. Bakt., Abt II, 49: 555-647, 1914. Brian, P. W.: Studies on the biological activity of griseofulvin, Ann. Botany 13: 59-77, 1949. Brian, P. W., P. J. Curtis, and H. G. Hemming: Glutinosin: a fungistatic metabolic product of the mould Mctarrhizium glutinosum S. Pope., Proc. Roy. Soc. (Lon- don), Ser. B, 135: 106-132, 1947. Buchanan, R. E., and E. I. Fulmer: Physiology and Biochemistry of Bacteria, Vol. II, the Williams & Wilkins Company, Baltimore, 1930. Bull, H. B. : Protein structure. Advances in Enzymol. 1: 1-42, 1941. BuRK, D., and C. K. Horner: Nitrogen assimilation by yeast, Wallerstein Labs. Communs. 2 : 5-23, 1939. BuRKHOLDER, P. R., and I. McVeigh: Growth of Phycomyces blakesleeanus in rela- tion to varied environmental conditions, Am. Jour. Botany 27: 634-640, 1940. *Cantino, E. C: The physiology of the aquatic Phycomycete, Blastocladia Pring- sheimii, with emphasis on its nutrition and metabolism. Am. Jour. Botany 36 : 95-112, 1949. Challenger, F., C. Higginbottom, and L. Ellis: The formation of organo-metal- loidal compounds by microorganisms. I. Trimethylarsine and dimethylethyl- arsine, Jour. Chem. Soc. 1933: 95-101. ESSENTIAL NON METALLIC ELEMENTS 113 Challenger, F., and H. E. North: The formation of organo-metalloidal compounds by microorganisms. II. Dimethylselenide, Jour. Chetn. Soc. 1934: 68-71. Dixon, M.: Manomctric Methods as Applied to the Measurement of Ceil Respira- tion and Other Processes, Cambridge University Press, New York, 1943. DoRRELL, W. W. : The oxidative respiration of Fusarium graminearurn, thesis, Uni- versity of Wisconsin, 1948. Dox, A. W. : The phosphorus assimilation of Aspergillus niger, Jour. Biol. Chem. 10 : 77-80, 1911-1912. *DuGGAR, B. M., and A. R. Davis: Studies in the physiology of the fungi, I. Nitrogen fixation, Ann. Missouri Botan. Garden 3: 413-437, 1916. Fellows, H.: The influence of oxygen and carbon dioxide on the growth of Ophio- bolus graminis in pure culture. Jour. Agr. Research 37: 349-355, 1928. Foster, J. W.: Chemical Activities of Fungi, Academic Press, Inc., New York, 1949. Fries, N.: X-ray induced parathio trophy in Ophiostoma, Svensk Botan. Tidskrift 40: 127-140, 1946. Fries, N.: Spontaneous physiological mutations in Ophiostoma, Hereditas 34: 338- 350, 1948. GoRTNER, R. A.: Outlines of Biochemistry, 1st ed., John Wiley & Sons, Inc., New York, 1929. GoRTNER, R. A.: Outlines of Biochemistry, 3d ed., John Wiley & Sons, Inc., New York, 1949. Gould, B. S., A. A. Tttell, and H. Jaffe: Biochemistry of Fusaria. The influence of diphosphopyridine nucleotide on alcoholic fermentation {in vivo), Jour. Biol. Chem. 146: 219-224, 1942. Haehn, H., and H. Leopold: Ueber eine Aspartasewirkung der Hefe, Biochem. Zeit 292 : 380-387, 1937. Harden, A.: .Alcoholic Fermentation, Longmans, Roberts and Green, London, 1932. Hawker, L. E.: The effect of vitamin Bi on the utilization of glucose by Melano- spora destruens Shear., Ann. Botany 8: 79-90, 1944. Herbst, R. M.: The transamination reaction, Advances in Enzymol. 4: 75-97, 1944. Hevesy, G. : Some applications of radio-active indicators in turnover studies, Advances in Enzyinol. 7: 111-214, 1947. Hockenhull, D. J. D.: The suphur metabolism of mould fungi: the use of "bio- chemical mutant" strains of Aspergillus nidulans in elucidating the biosynthesis of cystine, Biochim. et Biophys. Acta 3 : 326-335, 1949. *HoLLis, J. P.: Oxygen and carbon dioxide relations oi Fusarium oxysporum Schlecht. and Fusarium eumartii Carp., Phytopathology 38: 761-775, 1948. La Far, F.: Technical Mycology. Vol. II, Eumycetic Fermentation (trans. C. T. C. Salter), Charles Griffin & Co., Ltd., London, 1911. Leonian, L. H., and V. G. Lilly: Studies on the nutrition of fungi. I. Thiamin, its constituents, and the source of nitrogen. Phytopathology 28 : 531-548, 1938. Leonian, L. H., and V. G. Lilly: Studies on the nutrition of fungi. IV. Factors influencing the growth of some thiamin-requiring fungi. Am. Jour. Botany 27 : 18-26, 1940. Lilly, V. G., and L. H. Leonian: Nitrogen metabolism in Saccharomyces cerevisiae, Proc. West Va. Acad. Sci. 16: 60-70, 1942. LiNDEBERG, G. : Ucber die Physiologic Ligninabbauender Bodenhymenomyzeten, Symbolae Botan. Upsalienses 8(2) : 1-183, 1944. Mandels, G. R., and R. G. H. Siu: Rapid assay for growth: Determination of microbiological susceptibility and fungistatic activity. Jour. Bact. 60 : 249-262, 1950. 114 PHYSIOLOGY OF THE FUNGI *Mann, T.: Studies in the metabolism of mould fungi. I. Phosphorus metabolism in moulds, Biochem. Jour. 38: 339-345, 1944. Mann, T. : Studies in the metabolism of mould fungi. II. Isolation of pyrophos- phate and metaphosphate from Aspergillus niger, Biochem. Jour. 38: 345-351, 1944a. MiKSKY, A. E.: Chromosomes and nucleoproteins, Advances in Enzymol. 3: 1-34, 1943. NicKEKSON, W. J., and W. R. Carroll: The effect of culture history on the metabolic activities of cells of Zygosaccharomyces, Jour. Cellular Comp. Physiol. 22 : 21-32, 1943. NoRD, F. F., and R. P. Mull: Recent progress in the biochemistry of Fusaria, Advances in Enzymol. 5: 165-205, 1945. PiRSCHLE, K.: Biologische Beobachtungcn liber Hefewachstum mit besonders Berlicksichtigen von Nitraten als Stickstoffquelle, Biochem. Zeit. 218: 412-444, 1930. Plumlee, C. H., and A. L. Pollard: Studies on bromine-oxidizable sulfur-contain- ing compounds in mold metabolism, Jour. Bad. 57 : 405-407, 1949. Raistrick, H., and J. M. Vincent: Studies in the biochemistry of micro-organisms. 77. A survey of fungal metabolism of inorganic sulphates, Biochem. Jour. 43 : 90-99, 1948. Raulin, J.: Etudes chimiques sur la vegetation, Ann. sci. nat., Ser. V, 11: 93-299, 1869. Rippel, K.: Quantitative Untersuchungen iiber die Abhangigkeit der Stickstoff as- similation von der WasserstofRonenkonzentration bei einigen Pilzen, Arch. Mikrobiol. 2: 72-135, 1931. *RoBBiNS, W. J.: The assimilation by plants of various forms of nitrogen, Aj». Jour. Botany 24: 243-250, 1937. RoBBiNs, W. J., and V. Kavaxagh: Vitamin deficiencies of the filamentous fungi, 5otaM. i^e!;. 8: 411-471, 1942. ♦■Roine, p.: On the formation cf primary amino acids in the protein synthesis by yeast, Ann. Acad. Sci. Fennicae 26: 1-83, 1947. Rose, W. C: The nutritive significance of the amino acids, Physiol. Revs. 18: 109- 136, 1938. ScHEFFER, T. C, and B. E. Livingston: Relation of oxygen pressure and temper- ature to growth and carbon dioxide production in the fungus Polystictus versi- color, Am. Jour. Botany 24: 109-119, 1937. Semeniuk, G.: The dissimilation of glucose by Chaetomiuin funicola Cke. III. Some phosphorus relationships of Chaetomium funicola, Iowa State Coll. Jour. Sci. 18 : 325-308, 1943-1944. Siu, R. G. II., and G. R. Mandels: Rapid method for determining mildew suscepti- bility of materials and disinfecting activity of compounds. Textile Research Jour. 20: 516-518, 1950. Smith, V. M.: On the mechanism of enzyme action. XXXIX. A comparative study of the metabolism of carbohydrates, in the presence of inorganic and organic phosphates, by Merulius lacrymans and Marasmius chordalis, Arch. Biochem. 23 : 446-472, 1949. Steinberg, R. A.: Effects of barium salts upon Aspergillus niger and their bearing upon the sulfur and zinc metabolism of the fungus in an optimum solution, Botan. Gaz. 97: 666-671, 1936. Steinberg, R. A. : Growth of fungi in synthetic nutrient solutions, Botan. Rev. 5 : 327-350, 1939. ESSENTIAL NON METALLIC ELEMENTS 115 Steinberg, R. A.: Sulfur and trace-element nutrition of Aspergillus niger, Jour. Agr. Research 63: 109-127, 1941. Steinberg, R. A. : The process of amino acid formation from sugars by Aspergillus niger, Jour. Agr. Research 64: 615-633, 1942. *Steinberg, R. a. : Growth of fungi in synthetic nutrient solutions. II. Botan. Rev. 16 : 208-228, 1950. Stokes, J. L., IM. Gunness, and J. W. Foster: Vitamin content of ingredients of microbiological culture media, Jour. Bad. 47 : 293-299, 1944. Sumner, J. B., and G. F. Somers: Chemistry and Methods of Enzymes, 2d ed., Academic Press, Inc., New York, 1947. Tamiya, II.: Atmung, Giirung und die sich daran beteiligenden Enzyme von Aspergillus, Advances in Enzymol. 2 : 183-238, 1942. Tauber, H. : The Chemistry and Technology of Enzymes, John Wiley & Sons, Inc., New York, 1949. Ternetz, C. : Protoplasma bewegung und Fruchtkorperbildung bei Ascophanus carneus Pers., Jahrb. wiss. Botan. 35: 273-312, 1900. Ternetz, C: Ueber die Assimilation des atmospharischen Stickstoff durch Pilze, Jahrh. wiss. Botan. 44 : 353-408, 1907. Thorn, R. S. W. : The assimilation of nitrogen from amino acids by yeast, Jour. Inst. Brewing 43 : 288-293, 1937. TovE, S. R., H. F. Niss, and P. W. Wilson: Nitrogen fixation by the fungus, Phoma causarina, 49th meeting Soc. Am. Bact. (Abs. No. A-26, p. 59), 1949. Umbreit, W. W., R. H. Burris, and J. F. Stauffer: Manometric Techniques and Related Methods for the Study of Tissue Metabolism, Burgess Publishing Co., Minneapolis, 1945. VoLKONSKY, M. : Sur les conditions de culture et le pouvoir de synthese de Saprolegnia sp. Etude qualitative de I'alimentation carbon^e, azotee et sulfuric, Ann. inst. Pasteur 50 : 703-730, 1933. VoLKONSKY, M.: Sur I'assimilation des sulfates par les champignons: euthiotrophie et parathiotrophie, Compt. rend. acad. sci. 197: 712-714, 1933a. YoLKONSKY, M. : Sur la nutrition de quelques champignons saprophytes et parasites, Ann. inst. Pasteur 52: 76-101, 1934. Wilson, P. W. : The Biochemistry of Symbiotic Nitrogen Fixation, University of Wisconsin Press, Madison, 1940. WiRTH, J. C, and F. F. Nord: Essential steps in the enzymatic breakdown of hexoses and pentoses. Interaction between dehydrogenation and fermentation, Arch. Biochem. 1 : 143-163, 1942. Wolf, F. A., and F. T. Wolf: The Fungi, Vol. II, John Wiley & Sons, Inc., New York, 1947. Young, H. C, and C. W. Bennett: Growth of some parasitic fungi in synthetic culture media, Am. Jour. Botany 9: 459-461, 1922. CHAPTER 7 CARBON SOURCES AND CARBON UTILIZATION Carbon occupies a unique position among the essential elements required by living organisms. Almost half of the dry weight of fungus cells consists of carbon. Protoplasm, enzymes, the cell wall, and reserve nutrients stored within the cells are compounds of carbon. Carbon com- pounds are equally important in fugus nutrition. Fungi secure energy by oxidizing organic compounds. In addition to being the main struc- tural elements, carbon compounds play an equally important functional role. The number of carbon compounds known far exceeds the total of known compounds of all the other elements, because of the property of carbon of forming compounds in which carbon is linked to carbon in the form of chains and rings. Various other elements such as nitrogen, oxygen, and sulfur may serve as linking elements. While many carbon compounds are stable at ordinary temperatures, others are extraordinarily sensitive to a wide range of chemical reagents and to slight changes in the physical environment. Organic compounds differ in composition, structure, and configuration. These are key factors which must be considered in relation to utilization of organic compounds by fungi. Since more is known about carbohy- drates and related compounds as carbon sources, and about the manner in which they are dissimilated and assimilated, than about any other class of organic compounds, most of the discussion in this chapter will be devoted to these topics. In the main, only naturally occurring organic compounds will be considered. MONOSACCHARIDES AND RELATED COMPOUNDS The simple sugars, or monosaccharides, have the general formula C„(H20)n. The carbon chain is unbranched except in a few, very rare sugars. The functional groups present are primary ( — CH2OH) and secondary ( — CHOH — ) alcohol groups, and an aldehyde ( — CHO) or ketone ( — CO — ) group, actual or potential, is always present. The primary alcohol and aldehyde groups are restricted to the end positions of the carbon chain, while the ketone group is usually on the second 116 CARBON SOURCES 117 carbon in the chain. Sugars having an aldehyde group are called aldoses, those having ketone group, ketoses; the ending -ose denotes a sugar. In addition, the sugars are further classified according to the number of carbon atoms in the chain, e.g., pentoses, hexoses, or more specifically as aldopentoses, ketohexoses, etc. While it will be necessary in the discussion to follow to include some information about the chemistry and structure of the sugars, the reader is advised to consult suitable texts for further information. Those of Oilman (1943) and Pigman and Goepp (1948) are recommended. Compounds which have the same composition and the same molecular weight are called isomers. There are 16 aldohexoses (32, if the alpha and beta forms are considered), which have the same percentage com- position and the same functional groups as glucose (dextrose). There are eight possible ketohexoses isomeric with fructose. Two kinds of isomers exist among the sugars: First, there are those which have the same physical properties but differ in the direction in which they rotate plane-polarized light (enantiomorphs) . Isomers of this kind occur in pairs, and the configuration of the functional groups of one isomer is the mirror image of the configuration of the other. Enantiomorphs usually differ physiologically. One such isomer may be utilized and the other not, or one may be utilized much more rapidly than the other. Pasteur (1860) was the first to demonstrate that fungi are able to distinguish between such isomers. Penicillium glaucum utilized c?-tartrate more rapidly than Z-tartrate {d and / refer to optical rotation). Second, there are those isomers which, although they have the same functional groups, have these groups arranged in a different order, so that one isomer is not the mirror image of the other (diastereoisomers). It is usually safe to assume that one member of a pair of enantiomorphs will be better utilized than the other, but such an assumption about utilization of diastereoisomers is not possible. Since not all sugars of a group such as the aldohexoses are utilized by fungi, it is of interest to compare chemical structure or configuration with utilization. Not all fungi are able to utilize exactly the same sugars (Fig. 20). Whether a sugar is utilized or not depends upon both the configuration of the sugar and the particular abihties of the specific fungus. By configuration is meant the spatial arrangement of the hydrogen and hydroxyl groups. The long history of chemical investi- gation which established the configuration of the simple sugars must be passed by. Inasmuch as glucose is the key compound in sugar chemistry, as well as in physiology, particular emphasis will be devoted to this aldose. The structures of the glucose enantiomorphs are given at the top of page 119. 118 PHYSIOLOGY OF THE FUNGI A B CD Fig. 20. Growth of three fungi on four sugars. A, gkicose; B, fructose; C, sucrose; D, maltose. Top row, Monilinia Jructicola (8 days); middle, Mucor ramannianus (8 days); bottom, Ustilago striiformis, fragmenting strain (20 days). CARBOS SOURCES 119 1. 2. CHO H— C— OH 3. HO— C— H I 4. H— C— OH 5. 6. H— C— OH I CHoOH D-Glucose CHO I HO— C— H I H— C— OH I HO— C— H HO— C— H I CH.OH L-Ghicosc The letters d and l indicate that these sugars belong to different series; they do not indicate optical rotation. The small letters d and / have been used in the past to express two separate ideas, optical rotation or configuration. The use of d and I in the old literature makes it difficult at times to discover which enantiomorph was meant. The configuration of the secondary hydroxyl group farthest from the carbonyl group deter- mines to which series a sugar belongs. D-Glucose is the form which occurs naturally and is meant when glucose is used without qualification. Not all naturally occurring sugars belong to the d series; e.g., L-arabinose. For the sake of clearness and accuracy, the series designation should always be used where there is any chance of confusion and misinterpre- tation. Pigman and Goepp (1948) point out that only sugars of the galactose type occur naturally as both enantiomorphs. D-Galactose is fermented by some yeasts, while L-galactose is not. Hexoses. The following hexoses occur naturally: D-glucose, D-man- nose, D-galactose, L-galactose, D-fructose, and L-sorbose. It is doubtful if L-sorbose occurs in green plants, but it is formed from sorbitol by bacterial {Acetobacter suboxydans) oxidation (Bertrand, 1904). CHO CHO CHoOH H— C— OH HO— C— H C=0 HO— C— H 1 HO— C— H HO— C— H H— C— OH H— C— OH H— C— OH H— C— OH 1 H— C— OH H— C— OH CH2OH D-Glucose CH2OH D-Mannose CH2OH D-Fructose CHO 1 CHO CH2OH H— C— OH HO— C— H C=0 1 HO— C— H 1 H— C— OH HO— C— H HO— C— H 1 H— C— OH H— C— OH H— C— OH HO— C— H HO— C— H t CH2OH D-Galactose CH2OH ly-Galactose CH2OH L-Sorbose 120 PHYSIOLOGY OF THE FUNGI The configuration of glucose, mannose, and fructose is the same for carbons 3 to 6. In the presence of dilute alkali these sugars undergo enolization to produce the same enol form. D-Glucose CHO H— C— OH R D-Mannose CHO HO— C— H I R it CHOH II COH D-Fructose CH,OH C=0 I R R ±: Common enol form Other effects of alkali and heat on sugars were noted in Chap. 2. Many fungi will utilize these three sugars if configuration is important in determining availability. However, these sugars are not equivalent for all fungi. The fact that galactose is not utilized by all fungi which utilize the three closely related sugars is illustrated by the data in Table 22. Glucose is utilized by more fungi than any other sugar and is nearly a universal carbon source. In attempting to culture fungi of unknown nutritional requirements on synthetic or semisynthetic media, glucose should be the first carbon source used. However, there are a few fungi which are unable to utilize glucose, or any sugar, as a carbon source. Leptomitus lacteus (Schade, 1940; Scliade and Thimann, 1940) is unable to utilize glucose, fructose, galactose, or sucrose. Skoog and Lindegren (1947) have reported the behavior of 12 strains of Saccharomyces cere- visiae which did not utilize glucose when first isolated. These strains became adapted to glucose on sufficiently long exposure to this sugar. Cheo (1949) found certain isolates of Ustilago striiformis to be unable to grow on glucose when freshly transferred from a sucrose medium. After 2 to 4 weeks these isolates began to grow. This behavior suggests the formation of an adaptive enzyme which was not formed when these isolates were cultured on sucrose medium. Some fungi, such as L. lacteus, apparently lack the ability to form adaptive enzymes for glucose utili- zation and must be classed as absolutely incapable of glucose utilization, while the yeasts of Skoog and Lindegren and the isolates of U. striiformis are facultatively able to utilize glucose. The differences among these fungi probably lie in the ability to form adaptive enzymes. No carbon source can be utilized if the medium is lacking in any essential element or compound. Kinsel (1937) and Stevens and Larsh (1939) reported that Diplodia macrospora would grow only on disacchar- ides and not on media containing glucose or other monosaccharides. CARBON SOURCES 121 The explanation of this anomalous situation was given by Margolin (1940) and confirmed by Wilson (1942), who found that D. macrospora was deficient for biotin. It is probable that other vitamin-deficient fungi have been reported in the past as unable to utilize certain sugars owing to the absence of specific growth factors. Negative results reported in the literature are therefore to be viewed with caution. Wolf and Shoup (1943) studied the oxidation of carbohydrates by Allomyces arhuscula, A. javanicus, A. moniliformis, and A. cystogenus. All four species oxidized dextrin (degraded starch), while A. arhuscula oxidized maltose and sucrose in addition. The other common naturally occurring sugars, including glucose and fructose, were not oxidized. It has since been shown that A. arhuscula is deficient for methionine and thiamine (Yaw, 1950). While there is an immense amount of information scattered throughout the literature to the effect that a certain sugar is utilized by various species, much of this information deals with relatively few sugars. Critical studies on the utilization of the sugars are rare. Margolin (1942) studied the amount of growth of 21 fungi on four hexoses. These data (Table 22) were obtained under uniform conditions. A mixed nitrogen source (ammonium nitrate and amino acids) was used, and vitamins were supplied to the deficient fungi. The time chosen for harvest in this study was the time maximum weight was attained on glucose. This work suffers from the common defect that the yields are compared on the basis of a fixed time of harvest. The ideal way of determining the value of different sugars for fungi would be to study both the rate and amount of growth as a function of time of incubation. The following generalizations about utilization of the common hexoses may be drawn from the data in Table 22: (1) There is no single sugar which supports the maximum amount of growth for all of these fungi. (2) All of these fungi utilize glucose, although the maximum amount of growth was not always attained on this sugar. (3) The more closely the configuration of another sugar approaches that of glucose, the more fungi utilize it. It is believed that these generalizations are valid for all fungi which utilize sugars. Steinberg (1939) found D-glucose, D-fructose, D-mannose, L-sorbose, and sucrose to be equally effective in the nutrition of Aspergillus niger while D-galactose, lactose, glycerol, and mannitol were poor sources of carbon for this fungus. Herrick (1940) reported that two isolates of Stereum gausapatum grew on glucose, fructose, mannose, and galactose. One isolate made significantly better growth on fructose; the other grew equally well on all four sugars. This indicates that not all isolates of a species are alike in ability to utilize a given sugar. The utilization of different carbon sources by A. oryzae was investigated in detail by Tamiya 122 PHYSIOLOGY OF THE FUNGI (1932). This paper should he consulted for the experimental details and references to the literature. One hundred twenty-three carbon compounds were investigated, and of the hexoses, mannose supported Table 22. Milligrams of Mycelium Produced by 21 Fungi Grown on Media Containing Different Sugars AL the sugars were used at a rate which supphed 8 g. of carbon per liter. Each 125-ral. flask contained 20 ml. of medium. Cultures were incubated at 25°C. Each weight in the table is the average of 12 cultures. (Margolin, thesis, West Virginia University, 1942.) Fungus Blakeslea trispora Diplodia macrospora D. natalensis Fusarium lycopersici Helicostylum pyriforme Helmintiwsporum sativum. . . Mucor ramannianus Pilaira moreaui Phycomyces blakesleeanus . . . Phytophthora cactorum P. erythroseptica P.fagopyri Pythiomorpha gonapodyoides Pythium ascophaUon Rhizopus nigricans R. suinus Rosellinia arcuata Sordaria fimicola Syncephalastrum racemosum. Thielavia basicola Typhula variabilis Days of incu- bation 6 15 8 6 5 8 8 7 7 14 12 6 6 6 4 6 6 6 5 10 12 Mg. mycelium D- Glu- cose 91 83 199 108 126 75 89 40 138 119 79 89 152 85 121 130 73 121 131 60 181 D- Fruc- tose 94 55 154 101 81 128 118 32 130 40 20 51 122 56 114 128 58 162 141 54 122 D- Man- nose 98 71 89 100 126 83 115 45 139 16 81 19 79 84 117 136 49 147 126 55 113 D- Ga- lac- tose 123 55 50 126 99 46 116 44 74 11 17 11 14 10 121 135 33 28 140 78 23 Mal- tose 113 94 190 119 102 96 128 44 101 157 114 20 76 111 121 30 63 127 132 57 202 Suc- rose 10 58 199 74 11 100 12 11 111 77 93 130 142 116 7 12 38 16 15 61 126 Lac- tose 7 21 17 18 40 40 124 44 6 4 10 13 12 27 5 8 34 52 13 6 15 the most growth. Quantitative data on the utilization of L-sorbose by fungi is less abundant than for the other hexoses. Observations in this laboratoiy indicate that many fungi either do not utilize sorbose or do so slowly. Pentoses. The pentoses shown below occur naturally, mostly in the form of polysaccharides or other complex compounds. L-arabinose and D-xylose are the most easily available and have been more extensively used than the other pentoses. The formulas for the naturally occurring pentoses are given below: CARBON SOURCES CHO CHO 1 CHO HO— C— H H— C— OH H— C— OH 1 H— C— OH HO— C— H H— C— OH 1 H— C— OH 1 HO— C— H H— C— OH CH2OH D-Arabinose CH2OH L-Arabinose CH2OH D-Ribose CHO CH2OH 1 CHO H C OH 1 C— 0 H— C— OH HO— C— H H— C— OH 1 H— C— OH H— C— OH HO— C— H HO— C— H CH2OH D-Xylose CH2OH L-Xylulose CH2OH ir-Lyxose 123 Aspergillus niger utilizes D-xylose and L-arabinose but not their enantiomorphs, as is shown in Table 23. Many of the pentoses listed in Table 23 are difficult to obtain in quantity, which accounts for the varied amounts used per culture. Table 23. The Amount of Growth and Sporulation of Aspergillus niger on Various Pentoses Time of incubation, 4 days. Cultures incubated at 35°C. (Steinberg, Jour. Agr. Research 64, 1942.) Pentose Pentose, g. per culture Mg. mycelium Sporulation D-Lyxose D- Xylose L-Xylose D-Arabinose L-Arabinose D-Ribose L-Ribose 1.0 2.0 0.5 2.0 2.0 0.25 0.25 0.2 860.2 6.2 0 205.1 0 5.2 0 10 1 0 6 0 0 Herrick (1940) found Stereum gausapatum to utilize xylose better than arabinose, while Aspergillus oryzae utilizes arabinose better than xylose, (Tamiya, 1932). Lentinus lepideus utilizes xylose (Nord and Vitucci, 1947). A comparative study of five fungi on xylose and arabinose indi- cated that xylose was utilized either more completely or more rapidly than arabinose (Margolin, 1942). The data of Margolin are given in Table 24; for comparable growth of these species on other sugars, see Table 22. 124 PHYSIOLOGY OF THE FUNGI Methylpentoses. D-Isorhamnose, L-fucose, and L-rhamnose are related to D-glucose, L-galactose, and L-mannose in that carbon G with its primary alcohol group in these hexoses has been replaced by a methyl group. These methylpentoses have not been thoroughly investigated in nutri- tional studies involving many fungi. Aspergillus niger utilizes L-rham- nose to some extent, but L-fucose is not utilized (Steinberg, 1942). A. oryzae makes much poorer growth on L-rhamnose than on D-xylose or Table 24. Milligrams of IMycelium Produced by Five Fungi Grown upon Xylose and Arabinose These sugars were used at concentrations which suppHed 8 g. of carbon per liter. Each 125-ml. flask contained 20 ml. cf medium. Cultures were incubated at 25 °C. (Margolin, thesis, West Virginia University, 1942.) Fungus Blakeslea trispora Mucor ramannianus Phycomyces blakesleeanus . . . . Phytophihora erythroseptica . . Pythiomorpha gonapodyoides . Days of incubation 6 8 7 12 6 D-Xylose ' 77 77 126 15 33 L-Arabinose 49 74 85 7 18 * This sugar was called Z-xylose in the earlier literature. L-arabinose (Tamiya, 1932). Of the five fungi listed in Table 24 only Mucor ramannianus utilizes L-rhamnose (Margolin, 1942). Stereum gausapatum utilizes rhamnose about as well as arabinose (Herrick, 1940), Sugar alcohols. Reduction of the aldehyde or keto group of the simple sugars converts them into alcohols. Several sugar alcohols are widely distributed in nature. Only the formulas for three of the natu- rally occurring sugar alcohols will be given. CHoOH CHoOH 1 CH2OH H— C— OH HO— C— H H— C— OH [0— C— H HO C H HO— C—H 1 H— C— OH H— C— OH HO— C—H H— C— OH H— C— OH 1 H— C— OH CH2OH Sorbitol CH2OH Mannitol CHoOH Galactitol (Dulcitol) Most fungi appear to utilize the corresponding sugars with greater facility than the sugar alcohols. Data for the comparative growth of five fungi on these sugar alcohols and the parent sugars are given in Table 25. CARBON SOURCES 125 Sugar acids. Three types of sugar acids may be produced from aldoses by oxidizing the terminal groups. Oxidation of the aldehyde group yields aldonic acids, such as D-gluconic acid from glucose, while oxidation of the primary alcohol group yields uronic acids, such as D-galacturonic acid from D-galactose. Oxidation of both the aldehyde and primary alcohol groups yields saccharic acids. The uronic acids are widely distributed in natural polysaccharides such as plant gums and mucilages and in pectin. The fungi in nature must frequently come in contact with uronic acids, but data on utilization of these and other sugar acids are rare. Steinberg (1942) cultured Aspergillus niger on media which con- Table 25. Milligrams op Mycelium Produced by FrvE Fungi Grown upon Glucose, Mannose, and Galactose and the Corresponding Sugar Alcohols These compounds were used at a rate which supplied 8 g. of carbon per liter. Each 125-ml. flask contained 20 ml. of medium. Cultures were incubated at 25°C. (Mar- golin, thesis, West Virginia University, 1942.) Fungus D-Glu- cose Sor- bitol D- Man- nose Man- nitol D-Ga- lactose Galac- titol Blakeslea trispora Mucor ramannianus 90 89 138 79 152 12 93 59 10 13 98 115 139 81 79 9 149 108 8 9 123 116 74 17 14 10 6 Phycomyces blakesleeanus Phytophthora erythroseptica Pythiomorpha gonapodyoides . . . 6 8 10 tained 1 g. of the calcium salts of the following sugar acids per culture (the weight of mycelium in milligrams is given in parentheses) : 2-keto-D- gluconic (201), 5-keto-D-gluconic (25), D-gluconic (32), D-glucuronic (206), and mucic (102). Tamiya (1932) reports that A. oryzae utilizes D-gluconic acid. While such compounds as the sugar acids are little used in making media, they are of interest in attempting to discover the relation between structure and configuration on the one hand and utilization on the other. Mixed carbon sources. In nature the fungi usually come in contact with mixed carbon sources rather than a single source of carbon. Certain fungi make more growth when supplied with a mixture of carbon sources. This increased utilization may be expected only if one or both carbon sources are poorly utilized. Horr (1936) investigated the growth of Aspergillus niger upon mixtures of glucose and galactose. Some of these data are given in Table 26. If these two carbon sources were utilized independently, and without one affecting the utilization of the other, the weight of mycelium produced on the combination of 18 g. of galactose and 2 g. of glucose should be 42.4 -\- 145.6, or 188 mg. The actual yield was 577.4 mg. The experiment indicates that A. niger is able to 126 PHYSIOLOGY OF THE FUNGI utilize galactose to better advantage in the presence of glucose. The experiments of Steinberg (1939) on the effect of two poor carbon sources on the growth of A. niger were made at 35°C. Some combinations of poor carbon sources supported more growth than when these sources were used singly. Thus, the calculated weight of mycelium for the combination, D-mannitol-lactose was 21.4; the actual yield was 233.6 mg. Some combinations of poor carbon sources resulted in a decrease in amount of mycelium formed (glycerol-D-galactose : calculated yield, 243.7 mg.; actual yield, 154.7 mg.). The effect of mixed carbon sources in the amount of growth of Phy corny ces blakesleeanus and Pythiomorpha gonapodyoides appeared to be purely additive (Margolin, 1942). Table 26. The Effect of Galactose and Glucose, Singly and in Combination, UPON the Amount of Growth of Aspergillus niger Cultures incubated 7 days at 20°C. (Horr, Plant Physiol. 11, 1936.) Grams of Sugars Used per Liter Yield, Mg. per Culture 10 galactose 45 . 1 18 galactose 42.4 20 galactose 44.3 2 glucose 145 . 6 10 glucose 411 .0 18 galactose + 2 glucose 577.4 10 galactose + 10 glucose 1,151.6 All these results indicate that the effect of mixed carbon sources is highly specific. A mixture of poor carbon sources may or may not result in increased growth, depending on the carbon sources involved as well as the fungus concerned. The favorable effects of mixtures of poor carbon sources on the rate and amount of growth have been ascribed to the ease with which a fungus is able to synthesize certain key intermediates. If the synthesis of intermediate X from carbon source A is slow and difficult, and the syn- thesis of X is rapid from carbon source B, it is probable that growth will be more rapid on media which contain both carbon sources. ORGANIC ACIDS An organic acid is characterized by having one or more carboxyl ( — COOH) groups. Some organic acids are utilized as sources of carbon and in other ways. Two series of organic acids are especially interesting from the standpoint of physiology. The fatty acids are monocarboxylic acids; the higher members, when esterified with glycerol, form fats. The dicarboxylic acids, especially those which contain four carbon atoms, enter into the metabolic pathways of the fungi in various ways; e.g., utilization of ammonium nitrogen (Chap. 6). I CARBON SOURCES 127 The form in which an organic acid exists (free acid or salt) is a function of the pH of the medium or cells. The free acid is the predominant form at low pH values. The terms for an acid and its salt {e.g., fumaric acid, fumarate) are used in the literature somewhat loosely. The effect of a free acid and its anion may be different (Chap. 8). Leptomitus ladeus, w^hich does not utilize sugars, grows on various fatty acids — acetic, butyric to capric — but not on formic or propionic acids (Schade, 1940). Apodachlya hrachynema utilizes the same fatty acids as L. ladeus and also, fumarate, succinate and malate. Aspergillus niger, according to Steinberg (1942), makes some growth on acetate, lactate, tartrate, malate, and fumarate. Growth was very poor com- pared with that on sucrose. Dulaney (1949) reported that little strepto- mycin was produced when organic acids were used by Streptomyces griseus. Yeasts use acetate to synthesize fat (White and Werkman, 1947). Tamiya (1932) investigated the utilization of many organic acids by Aspergillus oryzae. Growth w^as poor on most of these compounds except quinic acid. While an organic acid may serve as the sole source of carbon for fungi, in general acids do not allow as much or as rapid growth as carbohydrates. An amino acid may serve as a source of both nitrogen and carbon. Peptone may serve as a source of carbon and nitrogen for many fungi. Aspergillus niger, when grown on peptone as the sole source of carbon, deaminates the peptides and amino acids and releases ammonia in quantities greater than the fungus can use. The utilization of amino acids as carbon sources by A. niger w^as investigated by Steinberg (1942a), who found certain combinations of "primary" amino acids to be utilized about three-fourths as efficiently as sucrose. The utilization of individual amino acids by Penicillium roqueforti and Fusarium oxysporum var. lycopersici was studied by Gottlieb (1946). Not all the naturally occurring amino acids were utilized as carbon sources by these fungi. The six-carbon straight-chain amino acids norleucine and lysine and the sulfur-containing amino acids cysteine and methionine were not utilized as carbon sources. Glycine and valine were poor carbon sources for P. roqueforti, while F. oxysporum var. lycopersici grew well on these amino acids. Alternaria solani, Helminthosporium sativum, Rhizoctonia solani, Fusarium moniliforme, Chaetomium globosum, and Aspergillus niger were unable to utilize the naturally occurring sulfur- containing amino acids as a source of carbon. Yeasts differ in ability to utilize different amino acids as the sole source of carbon (Schultz et al., 1949). Glutamic acid and proline were available to more species than other amino acids. It is characteristic of fungi cultivated on amino-acid media as the sole source of carbon that the medium becomes alkaline. This is probably due to accumulation of 128 PHYSIOLOGY OF THE FUNGI ammonia which results from deamination. In general, the amino acids appear to be poor sources of carbon. GLYCOSIDES The carbon sources to be discussed in this and the next two sections differ from those previously considered in that they undergo hydrolysis. The complex carbohydrates and carbohydrate-like compounds yield simple sugars w^hen hydrolyzed. In some instances, other compounds are also formed. In most instances, fungi utilize these compounds only after hydrolysis. Therefore, utilization will be dependent upon the pro- duction of the necessary hydrolytic enzymes. If a fungus is unable to perform this preliminary "digestion," such complex carbohydrates will be unavailable. Many of the compounds to be considered in this section are isomers. The simple sugars exist mainly in the form of ring structures, rather than the open-chain forms which were depicted in the previous sections of this chapter. The chemical evidence may be reviewed in Pigman and Goepp (1948) or other text dealing with the sugars. Glucose exists in aqueous solution as an equilibrium mixture of a-D-glucose and /3-D-glu- cose. These formulas contain a six-membered ring of w^hich one atom is oxygen (pyranose). Some sugars, however, contain a five-membered ring (furanose). The formulas for these two forms of glucose are given below: H OH HO H \ / \ / c , c- H— C— OH HO— C— H H— C— OH H— C— O— H— C— OH I HO— C— H I H— C— OH I H— C— O— CH2OH CH2OH a-D-Glucose /3-D-Glucose The simple glycosides are a widely distributed group of naturally occurring compounds which contain a sugar moiety and an alcohol or phenol moiety. The form glucoside was formerly used to designate com- pounds of this type irrespective of the sugar moiety. Specific glycosides are designated by adding the ending oside to the name of the sugar involved; e.g., glucoside, mannoside, etc. Two glucosides are formed when glucose is treated with methanol under appropriate conditions. The formulas are given below: CARBON SOURCES 129 H OCH3 H3CO H C , C- H— C— OH I HO— C— H I H— C— OH I H— C— O— H— C— OH HO— C— H H— C— OH I H— C— O— CH,OH CH2OH a-Methyl-D-glucoside /3-Methyl-D-glucoside These formulas correspond to the alpha and beta isomers of glucose. The proof that a-methylglucoside and a-glucose have the same structure was furnished by Armstrong (1903), who followed the enzymatic hydi'olyses of these glucosides polarimetrically. Our interest in the glycosides is not in the chemical structure per se, but in the fact that utilization of these and other compounds having the same type of glycoside linkage is dependent upon configuration. Differ- ent enzymes are required for the hydrolysis of the a- and /3-glycoside linkages. Some fungi possess both types of hydrolytic enzymes, others but one, and some fungi appear to lack both. Thus, certain yeasts ferment a-methylglucoside but not /3-methylglucoside. These yeasts have an enzyme or enzymes which catalyze the hydrolysis of the a-gly- coside linkage but not the /3-glycoside linkage (lactose-fermenting yeasts are able to hydrolyze ;S-glycosides). The use of the methylglucosides is not always a safe guide in pre- dicting which complex sugars will be utilized by fungi. Aspergillus niger utilizes /3-methylglucoside rapidly and completely, while a-methyl- glucoside is poorly utilized (Dox and Neidig, 1912). Attempts to adapt A. niger to utilize a-methylglucoside as a sole source of carbon were without much success, although the fungus apparently utilized this compound in the presence of sucrose (Dox and Roark, 1920). A. niger utilizes lactose poorly. Tamiya (1932) found A. oryzae made only a trace of growth on a-methylglucoside. /3-Methylglucoside was not tested. This fungus grows well on maltose. These results are, perhaps, not unexpected in view of the specificity of enzymes. The utilization of the naturally occurring simple glycosides by fungi has been investigated but slightly. OLIGOSACCHARIDES These sugars are derived from two, three, or four hexose sugars by the elimination of water. On hydrolysis, the individual sugars are regener- ated. Five factors which determine the structure of the oligosaccharides are (1) the component sugars; (2) the component sugar which functions 130 PHYSIOLOGY OF THE FUNGI as the alcohol; (3) the stereochemical nature of the glycoside linkage; (4) the carbon of the alcohol moiety which forms the glycoside linkage; and (5) the ring structure of the component sugars (see Oilman, 1943), Maltose. It is doubtful whether this disaccharide occurs free in nature. It is formed when starch is enzymatically hydrolyzed; on further hydrolysis two molecules of glucose are formed. This disac- charide is utilized by many fungi. The glycoside linkage is alpha in maltose. H OH C H-C-OH I HO-C-H I H-C-0 I H-C-0 I CH2OH Maltose Cellobiose. The occurrence of this sugar as a repeating unit in cellu- lose makes it important. Cellobiose differs from maltose only in the nature of the glycoside linkage. With few exceptions only fungi which produce enzymes which attack the /3-glycoside linkage will utilize this sugar. HO-C-H CH2OH CH2OH Cellobiose Since cellobiose and maltose differ only in the nature of the glycoside linkage, it would be interesting to compare the utilization of these two sugars by a large number of fungi. Cellobiose has been studied so infre- quently that the necessary data are lacking. CARBON SOURCES 131 Lactose. This sugar is probably present in the milk of all animals. Hydrolysis of lactose by acids or lactase yields a molecule each of glucose and galactose. This sugar is hydrolyzed by emulsin and is therefore a /3-glycoside. CH2OH Lactose Sucrose. This sugar is of common occurrence in plants. On hydroly- sis one molecule of glucose and one of fructose are formed; a mixed a- and j8-glycoside linkage unites the sugar moieties. Sucrose apparently is utilized by fewer fungi than maltose, but more extensively than lactose (see Table 22). CH2OH CH2OH Sucrose In addition to the three common disaccharides (maltose, lactose, and sucrose), many other oligosaccharides are known. Owing to cost and relative unavailability, these sugars have not been studied inten- sively. Some of these "rare" sugars are used in differential media in bacteriology. Brief mention will be made here of some of these sugars. The nonreducing disaccharide trehalose (mushroom sugar) is syn- thesized by various fungi and is fermented by many yeasts. Trehalose on hydrolysis yields glucose ; it differs from maltose in the position of the 132 PHYSIOLOGY OF THE FUNGI glycoside linkage. Tamiya (1932) reported that Aspergillus oryzae utilized trehalose and raffinose. The trisaccharide raffinose is obtained as a by-product of beet-sugar manufacture. On complete hydrolysis galactose, glucose, and fructose are formed in equivalent amounts. The structure for raffinose is given below. Volkonsky (1934) found raffinose to be utilized readily by Pythmm deharyanum and a species of Sporotrichum. One isolate of Phytophthora parasitica utilized raffinose rapidly, while another isolate utilized this sugar slowly. Phytophthora cactorum and P. palmivora utilized this sugar slowly. The great majority of fungi tested by Volkonsky did not utilize raffinose. CH2OH O— C ^ HO— C— H H— C— OH I H— C— O— H- H— C— OH HO— C— H I H— C— OH H— C— O— CHoOH D-Fructose CH2O- D-Glucose H— C H— C— OH I HO— C— H I HO— C— H H— C— O— CH2OH u-Galactose Raffinose Oligosaccharides and polysaccharides are utilized by fewer fungi than is glucose. All microorganisms which can utilize a given polysaccharide are also able to utilize its hydrolytic products (Van Niel, 1944). Not all polysaccharides yield glucose on hydrolysis, but the majority of them do. While the evidence at hand does not exclude the direct utilization of disaccharides by some fungi, it is probable that these sugars are hydrolyzed before utilization in most instances. Smith (1949) suggests that Marasmius chordalis attacks cellobiose by a route that involves neither preliminary hydrolysis nor phosphorylation. The failure of a fungus to utilize an oligosaccharide may be due either to the lack of the necessary hydrolytic enzyme or to inability to utilize the component sugars. Failure to synthesize the necessary hydrolytic enzymes appears to be by far the most common cause of nonutilization. This is borne out by the data in Table 22. Of the 21 fungi studied by Margolin, two failed to grow on maltose, while eight did not utilize sucrose. Since all these fungi grew well on glucose and fructose, it is evident that failure to utilize maltose and sucrose was due to the fact that these fungi could not hydrolyze these sugars. The nonutilization of lactose by Syncephala strum racemosum is evidently due to the failure of CARBON SOURCES 133 this fungus to synthesize lactase, for this fungus makes good growth on either glucose or galactose. The same argument applies to Blakeslea trispora, Fusarium lycopersici, Rhizopus nigricans, and R. suinus. Non- utilization of a complex carbohydrate is usually due to the lack of the necessary hydrolytic enzymes. The hydrolysis of oligosaccharides by fungi is easily demonstrated. Phycomyccs blakesleeanus utilizes sucrose while Mucor ramannianus does not. If the mycelium of P. blakesleeanus is removed from a flask of sucrose medium after several days' incubation and the flask reinoculated with M. ramannianus, the latter fungus will grow. P. blakesleeanus excretes sucrase, which catalyses the hydrolysis of sucrose to D-glucose and D-fructose, both of which are utilized by M. ramannianus. A complex carbohydrate and its hydrolytic products are not necessarily equivalent in all respects. Hawker (1947) reported that the amount of mycelium produced by Melanospora destruens was different when this fungus was grown on equivalent amounts of glucose, fructose, mixtures of glucose and fructose, and sucrose. More mycelium was produced from glucose than from an equivalent amount of sucrose, and this was true whether the concentrations of these sugars were low or high. On the other hand, perithecia were produced more abundantly on sucrose than on glucose media. Indeed, hydrolysis of the same lot of sucrose to glucose and fructose allowed the production of no more perithecia than other samples of these sugars. The conclusion seems inescapable that the particular structure of sucrose was in some way favorable for the pro- duction of perithecia. While a fungus may utilize an oligosaccharide and its hydrolytic products, it is not safe to assume that both are used with the same efficiency for all purposes. POLYSACCHARIDES The chemistry of the polysaccharides resembles that of the oligosac- charides except that the number of sugar residues is much larger. These substances constitute the bulk of carbohydrate materials synthesized by plants and animals. The most important polysaccharides are cellulose, starch, and glycogen. On hydrolysis simple sugars are formed. The molecular weights of polysaccharides may be very large; cellulose from different sources may have a molecular weight ranging from 200,000 to 400,000. The molecular weights of many polysaccharides are much less than that of cellulose. In general, polysaccharides are insoluble or only colloidally soluble. The utilization of these substances by fungi is dependent upon the excretion of the necessary hydrolytic enzymes. Pig- man and Goepp (1948) classify polysaccharides on the basis of the hydrolytic products as homopolysaccharides, which yield only one mono- saccharide on hydrolysis, and heteropolysaccharides, which yield two or 134 PHYSIOLOGY OF THE FUNGI more monosaccharides or related compounds on hj^drolysis. Cellulose, starch, and glycogen are members of the first class and yield glucose on hydrolysis. Polysaccharides are frequently named b}^ replacing the end- ing -ose of the parent monosaccharide by -an. Fructan (le\ailan) desig- nates a polysaccharide which yields fructose on hydrolysis. A hexosan is a polysaccharide which yields hexose sugars on hydrolysis, and a pen- tosan yields pentoses. Pectins are polymers of galacturonic acid. The heteropolysaccharides occur in lesser amounts than the homopoly- saccharides. Among them are the hemicelluloses, which on hydrolysis yield D-xylose as the principal sugar, the plant gums, and agar. Cellulose. Chemically, cellulose is a linear polymer of o-glucose. The glucose residues are joined together through /3-glycoside linkages as in cellobiose, and cellulose may be thought of as consisting of repeating cellobiose units. Norman and Fuller (1942) postulate that the majority of fungi are able to utilize cellulose. In spite of the importance of cellu- lose utilization by fungi in the economy of nature much remains to be learned about this process. It is commonly accepted that the first stage in utilization of cellulose is hydrolysis, although Campbell (1932) has suggested oxidation. The hydrolysis of cellulose may be expressed schematically as follows: cellu- lose — > cellodextrins -^ cellotetrose -^ cellobiose -^ D-glucose. Fungus cellulases appear to have been infrequently studied. Grassmann et al. (1933) separated cellulase and cellobiase from Aspergillus oryzae. This cellulase was inactive in hydrolyzing cellulose degradation products hav- ing a molecular weight less than 1,000 (six glucose residues), while the cellobiase hydrolyzed cellulose fragments containing from two to six glucose residues. Fungi differ widely in ability to utilize cellulose. In general, the rate of utilization of cellulose is less than that of glucose. This is probabty due to the insolubility of cellulose, which limits the action of cellulase to the surface, or to an inadequate rate of enzyme synthesis. The principal source of cellulose available to fungi in nature is wood and other plant remains, ^^^lile cellulose is the chief constituent in such materials, hemicelluloses, gums, tannins, and lignin are also present. The wood-rotting fungi have been classified according to whether they cause white or brown rots. The fungi which cause brown rots attack cellulose in preference to lignin. The fungi which preferentially attack the noncellulosic constituents of wood cause white rots. The latter species are apparently more numerous than those which cause brown rots. The following are some of the fungi listed by Nobles (1948) as causing white rots: Armillaria mellea, Ganoderma lobaturn, Lenzites hetulinus, Pleurotus ostreatus, Polyporus ahietinus, P. cinnabarinus, P. pargamenus. A few fungi causing bro^^^l rots are Daedalea quercina, Lentinus lepideus, CAR BOX SOURCES 135 Lenzites trabea, Merulius lacrymans, Polyporus betulinus, and Trametes americana. The effect of a typical fungus causing white rot on the composition of wood is given in Table 27. Polyporus pargamenus was allowed to act on blocks of aspen wood for 20 months. At the end of this time the wood block showed three degrees of attack. The tan-colored portion was altered least. The pink-colored portion was intermediate, while the white portion had lost the most lignin. P. pargamenus also degraded the cellulose somewhat, as shown by lower degree of polymerization. T.\BLE 27. The Effect of Polyporus pargamenus ix Altering the Composition of Aspen Wood Time of incubation 20 months. (Heuser et al., Arch. Biochem. 21, 1949. Published by permission of Academic Press, Inc.) Portion of wood block Lignin, % Pentosans, % CeUulose, % (calculated) Original Tan Pink \Miite 17.5 10.4 4.5 3.4 19.3 12.8 8.3 8.4 GO. 68 73.84 84.20 85.32 The effect of fungi causing brown rots on the composition of coniferous woods has been studied by Schubert and Xord (1950). Lenzites saepiaria in 13 months caused a decrease in cellulose in pine sawdust from 45.5 to 18.5 per cent. During this period the apparent lignin content increased from 33.9 to 50.1 per cent. Similar results were obtained with Lentinus lepideus and Poria vaillantii. For a recent review of the microbiological degradation of cellulose see Nord and Vitucci (1948). Starch. Like cellulose, starch is a polymer of D-glucose. The glucose residues are j oined through a-glycoside linkages, and starch (and glycogen) may be thought of as consisting of repeating units of maltose. Starch consists of two tj-pes of molecules. The linear portion of starch is called amylose, while the branched-chain fraction is known as amylopectin. Starch is sjTithesized by green plants, while glycogen is formed by animals and fungi. The enzymes which catatyze the hydrolysis of starch are known as amylases and were discussed in Chap. 4. The enzymatic hj'drolysis of starch may be represented schematically as follows : starch -^ dextrins— ^ maltose -^ D-glucose. The branched-chain dextrins are incom- pletely hydrolyzed by amylase, while the straight-chain dextrins are completely converted to maltose (]\Iyrback, 1948). Starch is insoluble in water. Only those furgi which produce amylase are able to utilize starch. This ability is common among fungi but not universal. Volkonsky (1934) found 26 isolates and species of the 136 PHYSIOLOGY OF THE FUNGI Saproliginales to utilize starch and its hydrolytic products (dextrin, maltose, and glucose). Thirteen other carbon sources, including fructose, were not utilized. Margolin (1942) found that 19 out of 21 fungi which utilized maltose also utilized dextrin. The nonutilization of starch by Sclerotinia libertiana has been suggested as the basis of a method of preparing potato starch (Kakeura, 1946). Few yeasts utilize starch, although maltose and glucose are readily utilized. All the fungi listed in Table 22 except Pythiwn ascophallon and Phy- tophthora jagopijri utilized dextrin. A comparison of the ability of fungi to utilize glycogen and starch has not been investigated thoroughly. Tamiya (1932) found the yield of mycelium of Aspergillus oryzae to be greater on glycogen than on dextrin. Dextrin was a better carbon source than starch. The role of the pectin-destroying enzymes in parasitism and the rotting of fruits and vegetables is discussed in Chap. 17. Presumably these fungi utilize some or all of the hydrolytic products of pectin (n-galacturonic acid and methyl alcohol). None of the fungi, in so far as is known, utilize agar as a source of carbon. A . niger utilizes the arabo-galactan from western larch as a source of carbon (Ratajak and Owens, 1942). HETEROTROPHIC UTILIZATION OF CARBON DIOXIDE The assimilation of carbon dioxide is not restricted to green plants. Carbon dioxide fixation has been demonstrated in bacteria, fungi, protozoa, liver slices, barley roots, and intact green plants in the absence of light. The basis for classifying organisms according to the way they utilize carbon dioxide is discussed by Werkman and Wood (1942), By the use of carbon isotopes an elegant method is available for demonstrating carbon dioxide assimilation. In addition, the mechanism of fixation can be studied. This involves isolation and degradation studies of the com- pounds synthesized Avhile the organisms were exposed to isotopic carbon dioxide. Either stable or radioactive carbon isotopes may be used. The finding of isotopic carbon in compounds synthesized is proof of assimilation. Aspergillus niger and Rhizopus nigricans were shown to assimilate car- bon dioxide (Foster et al., 1941). Radioactive carbon dioxide (C^i02) was used in these experiments. Mycelium of R. nigricans was suspended in 5 per cent glucose solution and agitated in a closed system containing isotopic carbon dioxide. At the end of the experiment the mycelium and the medium were analyzed for radioactivity. More than one-third of the carbon dioxide assimilated was incorporated into cell constituents which were not decomposed by boiling for 1 hr. with 2M hydrochloric acid. Carbon dioxide was assimilated under aerobic and anaerobic conditions. The data of such an experiment are given in Table 28. CARBON SOURCES 137 Table 28. Distribution of Radioactive Carbon (C^O in the Culture Medium AND Mycelium of Rhizopus nigricans Exposed to C^'02 in the Gas Phase for 30 Minutes Results are expressed as percentage of C^Oo assimilated. (Foster et ah, Proc. Natl. Acad. Sci., U.S. 27, 1941.) Substance tested Total C* in supernatant solution after removing cells. Fumaric * acid in this solution C* in neutral volatile distillate Total C* in acid extract of cells Fumaric * acid in this solution C* remaining in cells after acid extraction Aerobic 19 5 8 0 0 1 44 0 6 5 5 Anaerobic 29.0 25.0 0.2 30.0 12.0 41.0 * Designates radioactive carbon. It is probable that carbon dioxide enters into various metabolic proc- esses. Foster and Davis (1948) postulate that strains of Rhizopus nigricans which produce fumaric acid anaerobically do so according to scheme IV. Cantino (1949), in studying the metabolism of Blastocladia Scheme IV. A Scheme for the Anaerobic Transformation of Glucose into Fumaric Acid by Rhizopus nigricans* nC6Hi206 CH3CHOHCOOH Lactic acid I i +2H yf-nAU nCHsCOCOOH Pyruvic acid + CO2 HOOCCH2COCOOH Oxalacetic acid >- ■^ CO2+ CH3CHO Acetaldehyde + 2H CH3CH2OH Ethyl alcohol + 2H HOOCCHgCHOHCOOH Malic acid I * Courtesy of Foster and Davis, Jour. Bad., 56 : 335, 1948, & Wilkins Company. -HOH HOOCCH-.CHCOOH Fumaric acid Published by permission of The Williams pringsheimii, found that, by increasing the carbon dioxide in the gaseous phase, the formation of lactic acid was decreased, while the amount of 138 PHYSIOLOGY OF THE FUNGI succinic acid was increased. It was surmised that this fungus utilizes carbon dioxide, since none was set free. The formation of oxalacetic acid by the reaction between pyruvic acid and carbon dioxide suggests that heterotrophic carbon dioxide fixation may play a role in amino-acid synthesis. Support of this hypothesis may be found in the work of Ajl and Werkman (1949), who found the carbon dioxide requirement of Aerohacter aerogenes could be replaced by oxal- acetic, a-ketoglutaric, fumaric, or aspartic acid. For further information on carbon dioxide utilization by fungi see Foster (1949). UTILIZATION OF CARBON Carbon compounds are utilized by fungi for two general purposes, as a source of energy and as a source of the chief structural element. These two processes may be the same until a number of chemical transforma- tions have taken place but may then diverge after certain intermediate compounds are formed. The over-all use of carbon is quite easily determined, but it is a problem of a different order to trace all the chemical transformations which occur when a compound is utilized. Table 29. The Distribution of Carbon from Arabinose among the Products OF Metabolism of Fusarium lini (White and Willaman, Biochem. Jour. 22, 1928. Published by permission of Cambridge University Press.) Age of Mycelium, CO2, Alcohol, Lead pre- Sugar, Total culture, days % % % cipitate, % % carbon, % 5 0.8 0.6 7.6 90.6 99.6 10 3.4 4.4 7.6 0.6 85.2 101.2 15 4.6 6.1 6.6 1.0 80.4 98.7 25 4.0 9.4 3.3 1.5 81.2 99.4 40 10.4 20.8 9.9 1.7 55.2 98.3 Carbon balances. A general idea of the way a carbon source is utilized may be gained by following the amounts of mycelium synthesized, carbon dioxide evolved, and other metabolic products formed. If the initial amount of carbon is known, its distribution can be followed by analysis. From 95 to 99 per cent of the carbon is usually accounted for in such experiments. The accompanying data from White and Willaman (1928) illustrate this distribution of carbon from arabinose by Fusarium lini (Table 29). While the analytical difficulties in experiments of this kind are con- siderable, chemical analysis of the mycelium and the other metabolic products reveals how the carbon originally present in the carbon source is distributed. Such analyses are useful in detecting the major metabolic CARBON SOURCES 139 products. Carbon balances are especiall}^ useful in determining the effi- ciency with which a fungus produces metabolic products of value, such as alcohol and citric acid. For further examples see Raistrick ct al. (1931). Utilization ratios. The relations of the amounts of fungus metabolic products to the amount of carbon soiu'ce (or other substance) used are frequently expressed as ratios. However, these ratios are valid only for the fungi and the experimental conditions used. These ratios should be considered as absolute values only for the conditions under which they were obtained. The various utilization ratios are of less value than complete carbon balances, but the analytical determinations are fewer. To be of most value, these ratios should be determined at various intervals during incubation, because these ratios change with age. The most useful of these ratios is the economic coefficient, which is obtained by dividing the weight of mycelium and spores by the weight of sugar or other carbon source used. The residual carbon source in the medium must be determined at the end of an experiment. In general an efficient fungus will convert half the weight of sugar supplied in the medium into cellular material. The efficiency of most fungi when grown on laboratory media is much less. This is due in part to the use of unbalanced media and to the type of carbon metabolism taking place. The carbon which is not utilized for the synthesis of cellular material appears either as carbon dioxide or as intermediate metabolic products, such as alcohol and organic acids. In industrial applications it is desir- able to employ cultural conditions which divert a large part of the carbon used into the desired intermediate products, rather than into the produc- tion of mycelium and carbon dioxide. The economic coefficient of Fusarium sambucinum under various cultural conditions has been studied by Holzapfel (1925). This fungus utilized sucrose (0.33) and fructose (0.36) more efficiently than glucose (0.24). The economic coefficient varied with the concentration of the carbon source and w^ith the source of nitrogen, as w'ell as with the age of the cultures. For a discussion of other utilization ratios and examples, see Steinberg (1942), Peterson et al. (1922), White and Willaman (1928), and Fries (1938). Intermediary metabolism. The problem, to be considered here is the way fungi utilize the various sources of carbon available to them. From the data and discussion in the earlier part of this chapter it is clear that structure and configuration play an important role in determining which compounds may serve as a source of carbon for a given fungus. The availability of complex natural compounds, such as the carbohydrates, was found to depend upon the production of the necessary extracellular hydrolytic enzymes. The utilization of simple compounds, such as the 140 PHYSIOLOGY OF THE FUNGI monosaccharides, is likewise an enzymatically catalyzed chain of meta- bolic processes. It may be assumed that the chemical composition of the fungus will be about the same, irrespective of the carbon source utilized. Therefore, at some place along the path of synthesis the initial carbon sources are converted into the same compounds. It is probable that the original compounds are converted into the same intermediate compounds before synthesis. Thus, galactose is apparently transformed by Sac- charomyces fragilis into galactose- 1-phosphate, which is then converted into glucose- 1-phosphate (Caputto et at., 1949). These intermediate compounds then enter the various metabolic reaction chains which lead to the production of materials which make up the fungus. We may suppose that the first steps in utilization are those which transform a carbon source into key intermediates. The intermediate metabolic products should also serve as a source of carbon for the fungus in question. If a fungus transforms compound A into compound B, then compound B should serve as a source of carbon. Nonutilization of compound B indicates that this compound is not part of the metabolic pathway. This simple hypothesis neglects two important considerations : compound B may not enter the fungus cells with the same facility as compound A, or compound B may be toxic in the concentra- tions present. As an example of this approach, the work of Steinberg (1942) may be consulted. Since Aspergillus niger made only a trace of growth on D-gluconic acid, it seems probable that the first step in the utilization of glucose by this fungus is not the oxidation of the aldehyde group. The isolated enzymes from a fungus may also be studied to determine the reactions catalyzed, or the effect of specific enzyme inhibitors on the intact fungus may be studied. In some instances intermediates of sugar dissimilation are excreted into the medium and may be isolated. Thus, the production of acetaldehyde may be demonstrated by adding bisulfite to the medium. Acetaldehyde forms an insoluble addition product with this reagent. The excretion of intermediate metabolites may be due to slowness of the next step in the metabolic process. These products are usually utilized in the course of time. Among such intermediates which have been identified are acet- aldehyde, ethyl alcohol, and pyruvic acid. On the basis of the evidence now available we may not assume that all fungi utilize a sugar or other carbon source in exactly the same way, or that a fungus has only one metabolic pathway for the utilization of a sugar. Nord and Mull (1945) consider that species of Fusarium dis- similate carbohydrates by oxidation, by splitting the carbon chain, and by a phosphorylation mechanism. The relative importance of these three methods of attack depends upon the fungus involved and upon the environmental conditions. Identity of a metabolic product formed by CARBOX SOURCES 141 two fungi is not proof that the reaction mechanism is the same in both instances. Yeast and certain species of Fusarium produce alcohol, but the pathways from glucose to alcohol appear to be different. The mechanism of carbohydrate dissimilation by Fusarium lini, when grown upon a nitrate medium, is believed to take place as shown in scheme V. An essential feature of this scheme is the formation of pyruvic acid from both pentoses and hexoses. The intermediate steps in this biosynthesis by Fusarium lini have not been elucidated. A portion of the hydrogen derived from the dissimilation of carbohydrate is enzymatically trans- ferred and used for the reduction of nitrate ion which acts as a hydrogen acceptor. The nitrite produced inhibits the carboxylase enzyme system which transforms pyruvic acid into carbon dioxide and acetaldehyde. Pyruvic acid does not accumulate in the culture medium when ammonium nitrogen is used. Scheme V. The Pathway of Hexose and Pentose Utilization by Fusarium lini Grown on Nitrate Medium (Courtesy of Wirth and Nord, Arch. Biochem. \ : 155, 1942. Published by permis- sion of Academic Press, Inc.) Acceptor (Nitrate) Nitrite I Carboxylase system Hydroxylamine + ->- Pyruvic acid ^- Alcohol X reduction Amino acid Utilization Hexoses Pentoses Pyruvic acid is the key intermediate compound formed in the dis- similation of hexoses and pentoses by F. lini. The transformation of pyruvic acid into alcohol by F. lini and yeasts appears to follow the same pathway and to require the same coenzymes, cocarboxylase and code- hydrogenase I. The anaerobic dissimilation (fermentation) of glucose by yeast and the comparable process in muscle (glycolysis) have been intensively studied. These are perhaps the best understood of all metabolic proc- esses. Although it does not function in glucose dissimilation by F. lini in the same way as in yeast, phosphate plays a role in all these transforma- tions until pyruvic acid is formed. Many investigators have contributed 142 PHYSIOLOGY OF THE FLi.XGI to the scheme of gUicose dissimilation presented in schemie VI (Meyerhof, 1938, 1949). Further information about these reactions may be found in Sumner and Soraers (1947), Tauber (1949), and Prescott and Dunn (1949). Scheme VI. The Pathway of Glucose Dissimiliation by Yeast (Alcoholic Fermentation) and Muscle (Glycolysis) (Courtesy of Meyerhof, WaUerstein Labs. Communs. 12 : 256, 1949. Published by permission of WaUerstein Laboratories.) Glycogen, starch D-Glucose ± H,P04 II + H3PO4 Glucose- 1-phosphate (Cori ester)^=±GIucose-()-phosphate (Robison ester) Fructose-6-phosphate (Neuberg ester) + H3PO4 '4' Fructose-ljG-diphosphate (Harden- Young ester) Dioxyacetone phosphate* + H2 CHaOHCiOCHrOPOsHa /-Q^Glycero phosphate [CHsOHCHOH-CHaOPOsHs Glycerol + H3PO4 Aceta Ethanol dehyde + C02< Kd) 3-GIyceraIdehyde phosphate (Fischer-Baer ester) tCHOCHOHCH2-0-P03H2 + H2 + H3PO4 (d) 1,3-Diphosphoglyceric jicid + H3PO4 'CO-O-POsHsCHOH-CHa-O-POsH, (d) 3-Phosphoglvceric acid TCOOH-CHOH-CHa-O-POsHs (d) 2-Phosphoglvceric acid * CH2dHCH(OP03H2)COOH + H2O (Enol)-Phosphopvruvic acid TCH2:C(OP03Ho)(COOH) -Pyruvic acid + H3PO4 . ±H2 Lactic acid Pyruvic acid is a key intermediate compound in metabolism. Pyruvic acid serves as a source of carbon for many fungi, although the rate of growth on this substance is frequently slow. This is in accord with the hypothesis that intermediate metabolites are able to replace the original carbon source. The accumulation of this compound in the culture medium may be demonstrated by the formation of iodoform in the cold by adding a solution of iodine and making the medium strongly alkaline. The sensitive color test of Lu (2,4-dinitrophenylhydrazine) may also be used [see Friedemann and Haugen (1943) for details]. Acetaldehyde also yields iodoform under these conditions, but gentle heating will drive off CARBON SOURCES 143 this substance. AVe have noted in this laboratory that pyruvic acid ordinarily disappears from culture medium as the time of incubation is increased. The disappearance of the pyruvic acid in the culture medium is usually correlated with a rise in pH. Some typical reactions of pyruvic acid are shown in scheme VII. For a review of pyruvate metabolism see Stotz (1945). Scheme VIIo Some Typical Transformations of Pyruvic Acid CH3-CO— COOH H ^^ Pl7ri11M^ rtmA ^\ NH3+H CH3-CHOH-COOH Lactic acid 0 Pyruvic acid -CO2 CH3— CHO Acetaldehyde CH3-CH(NH2)-COOH ot - Alanine H CH3-COOH Acetic acid CH3-CH2OH Ethyl alcohol It is probable that most intermediates used in the synthesis of proto- plasm are synthesized from low-molecular-weight compounds. Acetate is used by yeasts for the synthesis of fats and other cellular constituents. Weinhouse and Millington (1947) studied the metabolism of isotopic acetate by yeast depleted of endogenous nutrients. Acetate was rapidly utilized. The distribution of the carbon from the isotopic acetate was Scheme VIII. Oxidation of Acetate by Yeast by Means of the Krebs Citric Acid Cycle* COOH Hooc— CH2— c— CHj— coon CH3— COOH -I HOOC— CHr— CO— COOH - T 2CH3— COOH HOOC— CH2— CHO H— COOH T HOOC— CH=CH— COOH T HOOC— CH2—C Ho— COOH <— HOOC— CH: -CO2 —CH— CHO H— COOH COOH CO2 HOOC— CH2—CH2— CO— COOH * Original scheme modified according to Weinhouse. Courtesy of Weinhouse and Millington, Jour. Am. Chem. Soc. 69: 3093, 1947. Published by permission of the American Chemical Society. determined by analysis. A portion of the acetate was oxidized; another portion was found in the lipide fraction and cell residue; some was con- verted to citric acid. It was calculated that from one-fourth to one-third of the lipides found in the yeast cells at the end of the experiment (a 7-hr. period) were newly synthesized from acetate. The cell residue (after extraction of the fats) contained only a little isotopic carbon. This is not surprising, since nitrogen was not furnished during these experiments. 144 PHYSIOLOGY OF THE FUNGI The mechanism of acetate oxidation by yeast is postulated by these authors to follow a modified Krebs citric acid cycle (scheme VIII). The oxidation of acetate is thus the result of a rather complex cyclic process. While the four-carbon dicarboxylic acids of the Krebs cycle are poor sources of carbon for most fungi, they are important in intermediary metabolism. These acids are readily interconvertible. The role of the keto acids in amino-acid synthesis was noted in Chap. 6. Lewis (1948) studied the metabolism of mutants of Neurospora crassa which were unable to synthesize either aspartic or glutamic acids. These amino Scheme IX. A Generalized Krebs Isocitric Acid Cycle Proposed to Illustrate the Pathways of Conversion of Carbohydrate into Aspartic and Glutamic Acids by Neurospora* Carbohydrate It Pyruvate -^ (CHg-CO-R) Cis-aconitate n Isocitrate A A oi -Ketoglutarate Oxalacetate Succinate * Courtesy of Lewis, Am. Jour. Botany 36: 294, 1948. acids could be replaced by a-ketoglutaric, succinic, malic, or fumaric acids. A generalized Krebs cycle was proposed by Lewis which indicates the pathway of synthesis of aspartic and glutamic acids from glucose (scheme IX). Compounds utilized by the Neurospora mutants are printed in italics. The probable location of the genetic block which prevents the biosynthesis of aspartic and glutamic acids is indicated by A. SUMMARY Organic compounds are utilized by fungi for the synthesis of structural and functional compounds and as sources of energy. The fungi utilize a wide range of natural organic compounds including those of great com- CARBON SOURCES 145 plexity. Not all fungi utilize all natural organic compounds, nor do all species utilize a given compound with the same facility. The composi- tion, structure, and configuration of organic compounds affect utilization, but the effect of these factors may be different for different fungi. The carbohydrates are the most common and important sources of carbon for the fungi. Sugars (and other compounds) having the same structure, but with mirror-image configuration, differ physiologically. Usually only one enantiomorph is utilized, or one is utilized much more rapidly than the other. Glucose is utilized by more fungi than any other sugar. Few fungi are unable to utilize glucose. A few species are appar- ently unable to utilize any sugar; e.g., Leptomitus lacteus. The species that utilize the pentoses, sugar alcohols, acids, and other simple organic compounds are fewer in number than those which utilize glucose. The oligo- and polysaccharides are utilized by fewer species than is glucose. The nature of the glycoside linkage as well as the sugar residues is important in determining whether these compounds are utilized by a given fungus. It is probable that most fungi hydrolyze oligosaccharides before utilization occurs. This does not exclude direct utilization in some instances. An oligosaccharide and its hydrolytic products are not always physiologically equivalent. The general order of availability of the three common disaccharides appears to be maltose, sucrose, and lactose. Among the polysaccharides, cellulose and starch are the most abundant. These compounds are insoluble and must be hydrolyzed or otherwise degraded to low-molecular-weight compounds before utilization. Only those fungi which form cellulase and amylase are able to utilize these compounds. This "digestion" is accomplished by enzymes. Ability to utilize other polysaccharides is also dependent upon possession of the necessary hydrolytic enzymes. Some fungi utilize carbon dioxide, but not as a sole source of carbon. It is postulated that carbon dioxide combines with pyruvic acid and other keto acids to form key intermediate products which are necessary for the formation of amino acids. The fate of the carbon supplied to a fungus is best determined by carbon-balance studies; i.e., by complete chemical analyses of the myce- livim and other metabolic products, including the carbon dioxide produced. The first step in utilization of sugars and other carbon sources is the formation of certain key intermediate metabolic compounds. These key compounds are in part utilized for synthesis and in part oxidized to pro- vide energy. The metabolic pathways leading to the formation of key intermediates differ, depending upon the environmental conditions and the fungus involved. Among the key intermediates pyruvic acid is especially noteworthy. Reduction of this compound yields lactic acid, while amination and reduction leads to alanine. Decarboxylation pro- 146 PHYSIOLOGY OF THE FUNGI duces acetaldehyde, which in turn may yield either ethyl alcohol or acetic acid. Acetate is utilized by yeast and other fungi for the synthesis of fats and other cellular constituents. A fungus utilizes a compound by a series of step-by-step transformation. Among the best understood of these metabolic activities is the transformation of glucose into alcohol by yeasts. REFERENCES Ajl, S. J., and C. II. Werkman: On the mechanism of carbon dioxide replacement in heterotrophic metaboHsm, Jour. Bad. 57 : 579-593, 1949. Armstrong, E. F. : Studies in enzyme action. I. The correlation of the stereoisomeric alpha and beta glucosides with the corresponding glucoses. Jour. Chem. Soc. 83 : 1305-1313, 1903. Bertrand, G.: Etude biochemique de la bacteria du sorbose, Ann. chim. et phys. 3: 181-288, 1904. Campbell, W. G.: The chemistry of the white rots of wood. III. The effect on wood substance of Ganoderma applanatum (Pers.) Pat., Fomes fomentarius (Linn.) Fr., Polyporus adustus (Willd.) Fr., Pleurotus ostreatus (Jacq.) Fr., ArmiUaria meUea (Vahl.) Fr., Trametes pit^i (Brot.) Ft., Sind Polystictus abietinus (Dicks.) Fr., Biochem. Jour. 26: 1829-1838, 1932. Canting, E. C: The phj^siology of the aquatic Phycomycete, Blastocladia Pring- sheimii, with emphasis on its nutrition u,nd metabolism, Am. Jour. Botany 36: 95-112, 1949. Caputto, R., L. F. Leloir, R. E. Trucco, C. E. Cardini, and A. C. Paladini: The enzymatic transformation of galactose into glucose derivatives, Jour. Biol. Chem. 179 : 497-198, 1949. Cheo, p. C: Stripe smut of blue grass (Ustilago striiformis forina Poae-pratensis): Spore germniation, artificial inoculation, pathological histology and growth in artificial media, thesis. West Virginia University, 1949. Dox, A. W., and R. E. Neidig: Spaltung von a- und /3-Methylglucosid durch Aspergillus nigcr, Biochem. Zeit. 46: 397-402, 1912. Dox, A. ^Y., and G. W. Roark, Jr.: The utilization of a/p/ia-methylglucoside by Aspergillus nigcr, Jour. Biol. Chem. 41: 475-481, 1920. Dulaney, E. L. : Observations on Streptomyces griseus. III. Carbon sources for growth and streptomycin production, Mycologia 41: 1-10, 1949. Foster, J. W. : Chemical Activities of Fungi, Academic Press, Inc., New York, 1949. Foster, J. W., S. F. Carson, S. Rl'ben, and IM. D. Kamen: Radioactive carbon as an indicator of carbon dioxide utilization. VII. The assimilation of carbon dioxide by molds, Proc. Natl. Acad. Sci. U.S. 27: 590-596, 1941. Foster, J. W., and J. B. D.wis: Anaerobic formation of fumaric acid by the mold Rhizopus nigricans. Jour. Bad. 56 : 329-338, 1948. Friedemann, T. E., and G. E. Haugen: Pyruvic acid. II. The determination of keto acids in blood and urine. Jour. Biol. Chem. 147: 415-442, 1943. Fries, N.: Ucber die Bedeutung von Wuchsstoffen flir das Wachstum verscheidener Pilze, Symbolae Botan. Upsalienses 3 : 2, 1938. GiLMAN, II.: Organic Chemistry, an Advanced Treatise, Vol. II, John Wiley & Sons, Inc., New York, 1943. Gottlieb, D. : The utilization of amino acids as a source of carbon by fungi, Arch. Biochem. 9: 341-351, 1946. Grassmann, W., L. Zeichmeister, G. Toth, and R. Stadder: Ueber den enzyma- tischen Abbau der Cellulose und ihrer Spaltprodukte. 2. Mitteilung iiber enzymatische Spaltung von Polysacchariden, J. Leibigs Ann. d. Chem. 503: 167-179, 1933. CARBOX SOURCES 147 ■*Ha\vker, L. E.: Further experiments on growth and fruiting of Melanospora destruens Shear in the presence of various carbohydrates, with special reference to the effects of gkicose and of sucrose, Ann. Botany (N.S.) 11: 245-260, 1947. Herrick, J. A.: The carbon and nitrogen metabolism of Stereurn gausapatum Fries., Ohio Jour. Sci. 40: 123-129, 1940. *Heuser, E., B. F. Shema, W. Shockley, J. W. Appling, and J. F. McCoy: The effect of lignin-destroying fungi upon the carbohydrate fraction of wood, Arch. Biochem. 21 : 343-350, 1949. HoLZAPFEL, II. H.: Untersuchung iiber die C-und N-Quellen einiger Fusarien, Cent. Bakt., Abt. II., 64: 174-222, 1925. ■*HoRR, W. II.: Utilization of galactose by Aspergillus niger and Pcnicillium glaucum, Plant Physiol. 11: 81-99, 1936. Kakeura, M.: Starches by using parasitic fungi, Japanese patent 172,381, Mar. 6, 1946. Chem. Abs. 43: 6850, 1949. KiNSEL, K.: Carbohydrate utilization by the corn Diplodias, Phytopathology 27: 1119-1120, 1937. Lewis, R. W. : Mutants of Neurospora crassa requiring succinic acid or a biochemi- cally related acid for growth, Am. Jour. Botany 35 : 292-295, 1948. Margolin, A. S.: The carbohydrate requirements of Diplodia macrospora, Proc. West Va. Acad. Sci. 14 : 56-59, 1940. Margolin, A. S. : The effect of various carbohydrates upon the growth of some fungi, thesis. West Virginia University, 1942. Meyerhof, O: The intermediary reactions of fermentation. Nature 141: 855-858, 1938. Meyerhof, O.: Glycolysis of animal tissue extracts compared with the cell-free fermentation of yeast, Wallerstein Labs. Communs. 12 : 255-264, 1949. Myrbach, K.: The structure of starch, Wallerstein Labs. Communs. 11: 209-219, 1948. Nobles, M. K.: Studies in forest pathology. VI. Identification of cultures of wood-rotting fungi, Can. Jour. Research, Sec. C, 26: 281-431, 1948. NoRD, F. F., and R. P. Mull: Recent progress in the biochemistry of Fusaria, Advances in Enzymol. 5: 165-205, 1945. NoRD, F. F., and J. C. Vitucci: On the mechanism of enzyme action. XXX. The formation of methyl-p-methoxycinnamate by the action of Lentinus lepideus on glucose and xylose. Arch. Biochem. 14: 243-247, 1947. NoRD, F. F., and J. C. Vitucci: Certain aspects of the microbiclegical degradation of cellulose. Advances in Enzymol. 8: 253-298, 1948. Norman, A. G., and W. H. Fuller: Cellulose decomposition by microrganisms. Advances in Enzymol. 2 : 239-264, 1942. Pasteur, L.: Note relative au Penicillium glaucum et a la dissymetrie mcleculaire des produits organiques naturels, Compt. rend. acad. sci. 51: 298-299, 1860. Peterson, W. H., E. B. Fred, and E. G. Schmidt: The fermentation of pentoses by molds, Jour. Biol. Chem. 54: 19-34, 1922. PiGMAN, W. W., and R. M. Goepp: Chemistry of the Carbohydrates, Academic Press, Inc., New York, 1948. Prescott, S. C, and C. G. Dunn: Industrial Microbiology, 2d ed., McGraw-Hill Book Company, Inc., New York, 1949. Raistrick, H., J. H. BiRKiNSHAW, J. H. V. Charles, P. W. Clutterbuck, F. P. Coyne, A. C. Hetherington, C. H. Lilly, M. L. Rintoul, W. Rintoul, R. Robinson, J. A. R. Stoyle, C. Thom, and W. Young: Studies in the bio- chemistry of micro-organisms. Trans. Roy. Soc. (London), Ser. B, 220: 1-367, 1931. Ratajak, E. J., and H. S. Owens: Optimal conditions for the hydrolysis of arabo galactan by Aspergillus niger, Botan. Gaz. 104: 329-337, 1942. 148 PHYSIOLOGY OF THE FUNGI ScHADE, A. L.; The nutrition of Leptomitus, Am. Jour. Botany 27: 370-384, 1940. ScHADE, A. L., and K. V. Thimann: The metabolism of the water mold Leptomitus lacteus, Am. Jour. Botamj 27: 659-670, 1940. Schubert, W. J., and F. F. Nord: Investigations on lignin and lignification. I. Studies on softwood lignin, Jour. Am. Chem. Soc. 72 : 977-981, 1950. ScHULTZ, A. S., D. K. Mc INI ANUS, and S. Pomper: Amino acids as carbon source for the growth of yeasts, Arch. Biochem. 22 : 412-419, 1949. Skoog, F. K., and C. C. Lindegern: Adaptation in a yeast unable to ferment glucose Jour. Bact. 53: 729-742, 1947. Smith, V. M.: On the mechanism of enzyme action. XXXIX. A comparative study of the metabolism of carbohydratqs, in the presence of inorganic and organic phosphates, by MeruHus lacrymans and Marasmius chordalis, Arch. Biochem. 23 : 446-472, 1949. Steinberg, R. A.: Relation of carbon nutrition to trace-element and accessory requirements of Aspergillus niger, Jour. Agr. Research 59 : 749-763, 1939. Steinberg, R. A.: The process of amino acid formation from sugars in Aspergillus niger, Jour. Agr. Research 64: 615-633, 1942. Steinberg, R. A.: Effects of trace elements on growth of Aspergillus niger with amino acids. Jour. Agr. Research 64: 455-475, 1942o. Stevens, N. E., and H. W. Larsh: Carbohydrate utilization by Diplodia macro- spora, Trans. Illinois State Acad. Sci. 32 : 82, 1939. *Stotz, E.: Pyruvate metabolism. Advances in Enzymol. 5: 129-164, 1945. Sumner, J. B., and G. F. Somers: Chemistry and Methods of Enzymes, Academic Press, Inc., New York, 1947. Tamiya, H.: Ueber die Verwendbarkeit von verschiedenen Kohlenstoffverbin- dungen im Bau- und Beitreibsstoffwechsel der Schimmelpilze. Studien iiber die Stoffwechselphysiologie von Aspergillus oryzae. IV. Acta Phytochim. (Japan) 6 : 1-129, 1932. Tauber, H.: The Chemistry and Technology of Enzymes, John Wiley & Sons, Inc., New York, 1949. Van Niel, C. B.: Recent advances in our knowledge of the physiology of micro- organisms, Bact. Revs. 8 : 225-234, 1944. VoLKONSKY, M.: Sur la nutrition de quelques champignons saprophytes et parasites, Ann. inst. Pasteur 52: 76-101, 1934. Weinhouse, S., and R. H. Millington: Acetate metabolism in yeast, studied with isotopic carbon, Jour. Am. Chem. Soc. 69: 3089-3093, 1947. ■*Werkman, C. H., and IL G. Wood: Heterotropic assimilation of carbon dioxide, Advances in Enzymol. 2 : 135-182, 1942. White, A. G. C, and C. H. Werkman: Assimilation of acetate by yeast. Arch. Biochem. 13:27-32, 1947. White, M. G., and J. J. Willaman: Biochemistry of plant diseases. X. Fermenta- tion of pentoses by Fusarium lini, Biochem. Jour. 22: 583-591, 1928. *WiLSON, W. E.: Physiological studies on two species of Diplodia parasitic on corn. Phytopathology 32 : 130-140, 1942. WiRTH, J. C, and F. F. Nord: Essential steps in the enzymatic breakdown of hexoses and pentoses. Interaction between dehydrogenation and fermentation, Arch. Biochem. 1: 143-163, 1942. Wolf, F. T., and C. S. Shoup : The effects of certain sugars and amino acids upon the respiration of Allomyces, Mycologia 35 : 192-200, 1943. Yaw", K. : Personal communication, 1950, CHAPTER 8 HYDROGEN-ION CONCENTRATION The growth of fungi and bacteria may be inhibited or prevented by media which are too acidic or too alkaline. A completely satisfactory medium may be made useless by the addition of relatively small amounts of strong acids or bases but may have its former usefulness restored if the excess acid or base is neutralized. This suggests that the ions which characterize acids and bases are particularly active in life processes. It is necessary to understand certain fundamental ideas about acidity and ways of measuring concentration of these ions before discussing in detail the effects of acids and bases on the activities of the fungi. IONIZATION OF COMPOUNDS Since water is the universal solvent for all life processes, our discussion will be confined to aqueous solutions. The chemical compounds which comprise natural and synthetic media may be divided into two classes, those which form ions in solution (acids, bases, and salts), and those which do not ionize (organic compounds in general, except organic acids and bases). Water is a compound of the first class, although it forms ions to a very slight degree. The ionization of water may be represented by the following equation: (1) HOH ^ H+ + OH- or HOH + HOH ^ H3O+ + OH" For each molecule of water ionized one hydrogen and one hydroxyl ion are formed. In any aqueous solution the product of the concentrations of the hydrogen and the hydroxyl ions (in moles) is equal to a constant (K^), Water is a neutral compound, i.e., the concentrations of hydrogen and hydroxyl ions are equal. A solution which contains a greater concentra tion of hydrogen than of hydroxyl ions is acidic ; a solution which contains a greater concentration of hydroxyl ions than of hydrogen ions is basic, or alkaline. Since all aqueous solutions contain hydrogen and hydroxy] ions, the deleterious effects of these ions must be due to their relative concentrations. At room temperature (23 to 25°C.) the concentration of hydrogen and hydroxyl ions in water is 1 X 10"^ mole per liter, or 1 mole each of these ions in 10 million liters. The degree of ionization of water increases with temperature. However, water is a neutral substance at 149 150 PHTSIOLOGT OF 71- 1 FVXGI all lempersTures because equal number? of hydrogen and hydrox\-l ions are pi^sent. The value of K^ at 23 to 2o^C. is obtained by multiphing the conceniraticais irf hydrogwi and hydroxyl ic«is present. (3j K, = : > :>-- 1 X icr- = 1 X lO-^* For ihe parpoee of this discussicsa. we will consider an acid to be a com- pound. whcKe aqueous scJuticHi contains a greater concentration of hydro- gen rhxn at hydroxyl ioais. A t-o^ is a compound whotse aqueous solution contains a greater concentraticai of hydro^l ions than of hydrc^n itms. Tbese deaniiioas vdll include a number ci compounds which are ordinarily con^doed as salts. A solmion of an acid c«Mitains a greater concentra- tion of hydrogen ions th.--^- pure water by Tirtue of the ionization of the acid. Hydrochloric acid ionises as follow?: C3) Ha ^ H- - a- In a sofaxtica of hydrochloric acid there are two sources ii C'f hy;h-oric. is considered to be completely itm- ixed even lu ; rated solutions- A weak acid, such as acetic, in IX s-i-u : I- - uly sli^tly about 1 per cent . The perc-entage of iooixi" : : i ^ increases as the dHuticm increases. The c<»- ccntri " : _ liS in. equal volumes of normal hydrochloric and acetic - - -^ — r chief differraice between these acids. Thus the stoaiiT r ' ~ ~ ' - -^pressed in tw^o ways. (1 < the total acidity A*^^ ■ . \ includes both the ionized and nonionized mofecuks CH iL i.e.. the titratab'ie acidity, and (2 ) the actwal acidity at any instant. - :_ ^i is a functicHi of the cc«icentration of hydrogen ions jwesBQt. T: - -tration c^ hydrogen ions is a nincrion of the con- centre I. - : i-izatitHi of the acid involved. It is the actual acadity ~ - esses. It is also necessary to consider the phy- . riieii- : ; z^ or cations which are associated wz- ^- - : -". i= impossibie to add just hydrogen 7 HZ MIAXC^G OF pH T: - ::. I. : UL : - _ ^-:i : ui in a soluticai can be expressed in Ti —ays. A derived unit pH is most used in biological work. The HYDROGES-IOS COSCESTRA TIGS' 151 calculated c .*• " t 1 1 / / i 1 1 1 A V ^ •^4--'fT y 1.0 0.8 0.4 Ml O.IN HCl 0.4 2.0 0.8 1.2 1.6 Ml. O.IN NAOH Fig. 21. Buffer-capacity curve.s of two media. The dotted line was obtained by titrating 20 ml. of glucose-asparagine medium with O.IjV hydrochloric acid and O.liV sodium hydroxide. The pH was determined after each addition of acid or base. The solid line was obtained in the same way on the above medium to which 10 mg. of neutralized glutamic acid had been added, (Courtesy of Robbins and Schmitt, Am. Jour. Botany 32 : 324, 1945.) buffer one unit wdll depend upon the concentrations of the buffer acid and salt present. The hufer capacity of a medium is measured by titrating a definite volume of medium (usually 100 ml.) with standard acid and alkali. The pH is measured after each addition of acid or alkali. From the curve drawn from these data the buffer capacity for any range of pH A^alues may be obtained. The curves in Fig. 21 illustrate the buffering capacity of two media (Robbins and Schmitt, 1945). These media differed in the buffers present. The unsymmetrical nature of the curves is due to the presence of overlapping buffers. The pH of culture media may be controlled within desirable limits, in some instances, by adding calcium carbonate to the medium. Calcium carbonate is essentially insoluble in neutral and alkaline media but acts as a neutralizing agent for acids. The calcium carbonate is used up as acid is produced by a fungus. The degree of neutralisation achieved depends upon the amount of calcium carbonate added and whether the 156 PHYSIOLOGY OF THE FUNGI cultures are agitated. See Foster (1949) for a discussion of the use of calcium carbonate in industrial microbiological processes. For fungi which have an extremely narrow pH range, the special cul- ture flask devised by Cantino (1949) for culturing Blastodadia pring- sheimii may be used (Fig. 22). A base (or acid) is placed in the side arm and an internal indicator of the desired pH range is added to the medium. A little of the base is tipped into the culture flask as desired. Flasks with two side arms may be used so that either acid or base maybe added to the culture medium. Fig. 22. Flask designed for the study of glucose dis- similation by Blastodadia. A, the side arm containing NaOH for neutralization; B, sintered-glass aerator; C, inlet for aeration with differ- ent gas mixtures; D, the outlet for removal of media. (Courtesy of Cantino, Am. Jour. Botany 36 : 100, 1949.) METHODS OF DETERMINING pH VALUES Only two general methods of measuring pH values will be discussed. The colorimetric method is simple, inexpensive, and sufficiently accurate for most purposes, but it cannot be used with highly colored or turbid media. The potentiometric method using the glass electrode is more accurate and is often the preferred method. Colorimetric methods. The use of indi- cators w^hich change color in response to varying concentrations of hydrogen ion is the basis of this method. Indicators may be considered as weak acids or bases, and as such they act as buffers, but the amounts used are so small they do not affect the accuracy of a determination. For methods of measuring the pH of unbuffered solutions see Snell and Snell (1948). The property of these indicator buffers which distinguishes them from other buffers is that the colors of the salts and free acids or bases (nondissociated) are different. Within the usable pH range, the color of the indicator is a function of the hydrogen-ion concentration of the medium. For exam- ple, bromocresol purple (p/va, 6.3) is yellow in solutions having pH values of 5.2 or less and purple at pH 6.8 or more. Within the pH range 5.2 to 6.8 the color changes from yellow to purple. To determine the pH value of an unknown solution within this range, the indicator is added to equal amounts of standard buffers and the unknown solution, and from the color of the standard buffers of known pH, the pH value of the unknown may be estimated to within 0.1 pH unit. By a suitable choice of indicators the pH range of interest may be covered. A few indicators with their pH ranges are listed in Table 32. Two methods of color comparison are in general use. The first involves HYDROGEN -ION CONCENTRATION 157 the use of the familiar comparator block. A slight color or turbidity of the medium may be compensated for by the use of suitable blanks. A porcelain spot plate may be used instead of a comparator block with considerable saving of time and materials, although the accuracy is some- what less. Drops of the indicator are added to the depressions in the spot plate. A drop of the medium is added to one depression, and drops of standard buffers to the other depressions. The pH of the medium is estimated from the pH of the buffer which yields a color matching that developed in the medium. Table 32. The pH Range and Color Changes of Various Indicators (Courtesy of Eastman Kodak Company.) Indicator Bromophenol blue . Bromocresol green. Chloro phenol red . . Bromocresol purple Bromothymol blue . Phenol red pH range 3.0-4.7 3.8-5.4 4.8-6.8 5.2-6.8 6.0-7.6 6.8-8.4 Color change Yellow-blue Yellow-blue Yellow-red Yellow-purple Yellow-blue Yellow-red All colorimetric methods of measuring pH require the use of standard buffers (buffers of known pH) or permanent standards. Buffers may be prepared in the laboratory or purchased from laboratory supply houses. It is convenient to use prepared buffer tablets, which need only to be dissolved in a measured amount of water before use. Potentiometric pH meters also require the use of a standard buffer for calibration. The easiest of these to prepare is a saturated solution of potassium hydro- gen tartrate (pH 3.57). The use of this buffer was recommended by Lingane (1947). It is simple to prepare, and temperature affects the pH very little. From Table 32 it will be noted that the pH range of a single indicator is less than two pH units. Much time can be saved in pH determinations by the use of a wide-range indicator to determine the approximate pH before using a single indicator for the final measurement. Wide-range indicators (pH range 2 to 10) may be purchased or prepared by mixing suitable indicators (Snell and Snell, 1948). The pH value of a medium may easily be determined within 0.5 pH unit by the use of a wide-range indicator. Either the comparator block or the spot-plate method may be used. For detailed information about indicators, see Kolthoff and Rosenblum (1937). Potentiometric methods. The potential difference which develops between certain pairs of electrodes when they are dipped into a solution is a function of the hydrogen-ion concentration. Solutions which give 158 PHYSIOLOGY OF THE FUNGI rise to the same potential difference have the same pH value. Modern pH meters are calibrated in pH units so that direct readings are obtained. Color or turbidity does not affect potentiometric measurement of pH. The glass electrode in conjunction with the calomel half cell is the most commonly used for liquids of biological interest. The glass electrode consists of a bulb blown from a special glass. The bulb is filled with O.IA'^ hydrochloric acid. A potential difference develops between the inside and the outside of the electrode; the magnitude of this potential difference depends upon the hydrogen-ion concentration of the liquid in which the bulb is dipped. Measuring the potential difference which develops between the glass electrode and the calomel half cell is equivalent to determining the pH value of the unknown solution. Sensitive auxiliary electrical equipment is required to measure this potential difference. For a discussion of the glass electrode, see Dole (1941). Many suitable pH meters are available. The trend appears to be toward instruments which use alternating current rather than batteries as a source of power. Since the details of operation are somewhat dif- ferent for the various makes, the directions of the manufacturer should be consulted. The pH of media should be determined before autoclaving and the reaction adjusted by the addition of acid or alkali if necessary. The pH of a sample of a medium should also be determined after autoclaving and before inoculation. The pH value at this time is known as the initial pH. Alkaline media absorb carbon dioxide from the atmosphere, causing a slow decrease in pH, Pritham and Anderson (1937) reported that the pH of uninoculated alkaline media may decrease as much as two units during the course of an experiment. This factor is of particular impor- tance when upper pH limits are being investigated. For methods of adjusting pH, see Suggested Laboratory Exercises. EFFECTS ON FUNGI Hydrogen and hydroxyl ions are present in all media and in substrates upon which fungi grow in nature. The pH of the medium exerts a decided effect upon the rate and amount of growth and many other life processes. A medium may have a pH which is favorable for growth and unfavorable for sporulation or other processes. The production of pig- ments, vitamins, and antibiotics may be influenced by the pH of the medium. As a result of metabolic activity a fungus ordinarily changes the pH of the medium upon which it grows. pH limits. The upper and lower pH values between which a fungus grows form the pH range of that species. The pH values which inhibit growth vary with the species. Between the limiting pH values there is a pH range which allows optimum growth. An initial pH of 5 to 6 is HYDROGEN-ION CONCENTRATION 159 satisfactory (not necessarily optimum) for the majority of the fungi. The optimum pH ranges for Blastocladia pringsheimii, Allomyces arhus- cula, and Blastocladiella simplex are rather narrow (Emerson and Cantino, 1948) (see Fig. 23). Most of the pH optima reported in the literature are less than 7. Meacham (1918) reported pH 3 to be optimum for Lenzites saepiaria, Fomes roseus, Merulius lacrymans, and Coniophora cerebella. Wolpert (1924) found the pH optimum of various Basidio- mycetes to be in the neighborhood of 5.5. Johnson (1923) reported that the upper pH limit of Penicillium varidbile is 10.1 to 11.1, which is con- Blastocladfa Allomyces Blastocladiella Fig. 23. Relation of pH of the medium to growth of Blastocladia -pringsheimii, Allomyces arbuscula, and Blastocladiella simplex. (Courtesy of Emerson and Cantino. Am. Jour. Botany 35: 162, 1948.) siderably higher than that of most fungi. The lower pH limits reported vary from 5.3 for B. simplex (Emerson and Cantino, 1948) to 0.5 for Acontium velatum and an unidentified imperfect fungus (Starkey and Waksman, 1943). The method used to determine the pH limits of a fungus is to inoculate a series of nutrient solutions having pH values spaced 0.2 to 0.4 unit apart. Growth may be observed visually, or the mycelium may be weighed. Such media should be well buffered. The pH limits for a given fungus as determined in different laboratories are frequently at variance. This is not unexpected, since the composition of the medium and the nature of the buffer influence the tolerance of fungi to hydrogen and hydroxyl ions. The behavior of Marasmius graminum is revealing (Lindeberg, 1944). Calcium ion was effective in overcoming the toxic effect of an initial pH of 3.3. The weight of M. graminum after 12 days was 0.4 mg., but when calcium sulfate was added to the medium, the yield was 8.0 mg. Tamiya (1928) also found calcium ion to protect Aspergillus oryzae to some extent against high concentrations of hydrogen ion. The optimum pH for Gibber ella saubinetti is lower when calcium is present in the medium (Lundegardh, 1924). Wolpert (1S24) also found 160 PHYSIOLOGY OF THE FUNGI the pH range of many fungi to vary on different media and concluded that the widest pH range was obtained on favorable media. The temperature of incubation may influence the optimum pH as well as the pH range of a fungus. The optimum pH for Phacidium infestans is 4.5 at 5°C., 5.0 at 10°C., 5.5 at 15°C. and 6.0 at 20°C. (Pehrson, 1948). The pH range of Armillaria mellea on a sucrose-peptone medium was reported to be 2.5 to 7.5 at 15°C., 2.0 to 7.8 at 25°C., and 2.5 to 7.4 at 35°C. (Wolpert, 1924). 21 0 180 150 .120 ■o .^ 90 o 60 30 .y^""^* ^^^0--^ — 1 ^ -.• / ,.-'-' ^/-'- ■'"/ ucose-). oofossiui 77 nitrate \ 7 ^ — ^Gi \ \ \ \ / > ^x ST — \ um sulfa 1 X /^ lucose-ammoni fe £. xJ 90 8.0 7.0 60 5,0 4.0 3.0 20 25 0 5 10 15 Doys of incubation Fig. 24. Rate of mycelial growth of Sordaria fimicola and accompanying changes in pH of two media. Media contained biotin but no thiamine. Sohd hnes indicate growth, and the broken lines represent pH values. Two pH optima have been reported for a number of fungi. Rhizopus nigricans, when grown on potato-glucose liquid medium, has two optimum pH ranges, one on either side of the isoelectric point of the mycelium, which was about pH 5.5 (Robbins, 1924). Scott (1924) reported Fusarium lycopersici to have two optimum pH ranges for growth on glucose-nitrate medium: pH 4.5 to 5.3 and 5.8 to 6.8. Mathur et al. (1950) obtained evidence that there are two optimum pH ranges for the sporulation of Colletotrichum Undemuthianuni. In addition to the use of media having low initial pH values, the lower pH limit may be determined in some instances by choosing a medium in which the fungus produces sufficient acid to inhibit growth completely. This is illustrated by the pH and growth curves of Sordaria fimicola in Fig. 24. This fungus was grown upon a glucose-ammonium sulfate medium having initial pH 6.0; after a few days the pH of the culture medium fell to 3.3 and remained there for 5 weeks. More difficulty may be experienced in determining the upper pH limit. If a fungus is able HYDROGEN-ION CONCENTRATION 161 to make a trace of growth in an alkaline medium, the carbon dioxide produced will lower the pH. Organic acids may also be produced. Car- bon dioxide from the air will be absorbed by alkaline media. 0.08 c "e t 0.06 0) 0) a. ■I 0.04 _>i 2 •o >« o % 0.02 •a m a. ' ' Urease ^ \ \ ■J 7 / .f N 7 pH Fig. 25. of urea 25. The shift of optimum pH for urease activity due to change in concentration sa. (Courtesy of \'an Slyke, Advances in Enzymol. 2: 41, 1942. Pubhshed by permission of Interscience Publishers, Inc.) pH Pig. 26. The effect of pH on the rate of linear growth of Neurospora crmsKi. tesy of Ryan, Beadle, and Tatum, Am. Jour. Botany 30: 790, 1943.) (0,11. 1 V It was pointed out in Chap. 4 that pH affects the activity of a.i/jymes. In general, there is a striking correlation between the optimum pH range for most enzymes and the optimum pH range for gro^vth. In a survey of the literature Haldane (1930) found all but 9 of 105 enzymes to have optima between pH 4 and 8. Most fungi have pH optima for growth ief2 PHYSIOLOGY OF THE FUNGI between these limits. The effects of pH upon the activity of urease (Van Slyke, 1942) and upon the rate of growth of Neurospora crassa (Ryan et al., 1943) are shown in Figs. 25 and 2G. From the general similarity of these two curves it appears probable that pH affects the rate of growth of fungi, at least in part, by modifying the rate of certain enzymatic reactions. pH changes in media during growth. Fungi, as a result of their metabolic activities, ordinarily change the pH of the media in which they grow. These changes cannot be studied by making a single deter- 8.0r 8 10 2 3 4 5 6 7 Doys of incubation Fig. 27. Changes in pH during incubation of Sordaria fimicola in different volumes of liquid glucose-casein hydrolysate medium at 25°C. mination of pH at any fixed time. Just as it is necessary to study growth as a function of time of incubation (growth curve), it is necessary to deter- mine the pH changes in an inoculated medium day after day to obtain a complete representation of these changes (pH curve). The pH of the medium should be followed in connection with the other functions being studied. Since fungi differ in their metabolic activity and rate of growth, the pH changes produced in the culture medium will differ. The pat- terns of pH changes for the same fungus will depend upon the composition and concentration of the media used. As an illustration of the effect of the composition of the medium upon the pH changes, some of our data for Sordaria fimicola are given in Fig. 24. The correlation of the pH changes with the rate and amount of growth of this fungus may be obtained by comparing the growth curves obtained at the same time. From Fig. 27 it is evident that the hydrogen- HYDROGEN-ION CONCENTRATION 163 ion concentration of a nutrient solution may change 10,000-fold during a few days as a result of the metabolic activities of a fungus. These changes in pH are due to changes in the relative amounts of acids and bases formed or withdrawn and to the ionization constants of these compounds. Some of the metabolic processes which result in a change in pH of a nutrient solution are discussed below. The utilization of cations, such as ammonium ion, for the synthesis of protoplasm or for any other purpose whereby essentially non-ionic com- pounds are formed, leaves an equivalent number of anions in the nutrient solution. Since solutions are electrically neutral, an equivalent number of both cations and anions must be present. Thus, when an equivalent of ammonium ion is transformed into non-ionic compounds, an equivalent of some other cation or cations will be formed in the nutrient solution. These ''new" cations are usually hydrogen ions, which are formed from water. If it is assumed that both cations and anions are adsorbed on the protoplasmic membrane, the process may be thought of as replace- ment. The production of acid would result from the utilization of other cations as well. The utilization of nitrate ion or other anion such as phosphate or sulfate for the formation of non-ionized compounds has the effect of increasing the hydroxyl-ion concentration of the medium. We may assume the same type of mechanism as before, except that the anion released to the nutrient solution is the hydroxyl ion. Fungi produce acids from nonacidic nutrients such as carbohydrates. Among these acids are carbon dioxide and various organic acids such as pyruvic, citric, and succinic acids. Carbon dioxide combines with water to form carbonic acid, which is unstable in the presence of stronger acids and decomposes to set free carbon dioxide. Under alkaline conditions carbonic acid reacts with bases to form bicarbonates. Pyruvic acid accumulates in the nutrient solution in which many fungi are grown, and in some instances the formation of this acid accounts for a considerable part of the early depression of pH. The eventual utilization of pyruvic acid causes the pH of the nutrient solution to rise. Other metabolizable acids behave similarly. Ammonia is, perhaps, the most common basic substance produced by fungi. Piricularia oryzae produces ammonia in considerable amounts (Henry and Andersen, 1948). The production of ammonia results from the deamination of amino acids and proteins. The processes discussed above may occur simultaneously. Whether a culture solution becomes more acid or alkaline depends upon the extent of these various processes. In general, the processes which produce acid pre- dominate during early growth, especially when ammonium nitrogen is used. The importance of the composition of the medium in determining what 164 PHYSIOLOGY OF THE FUNGI changes in pH will take place during growth is illustrated by the work of Dimond and Peltier (1945), who studied the pH changes produced by Penicillium notatum as a function of the carbon and nitrogen nutrition. When the initial pH was 6.0 and sodium nitrate was the nitrogen source, the lowest pH values attained on different sugars were glucose, 5.1; sucrose, 4.0; lactose, 3.2; maltose, 4.8; and galactose, 4.8. These were the lowest pH values attained under these conditions. In another experi- ment a mixture of tryptophane, asparagine, and cystine w^as used as the nitrogen source. The pH again varied with the sugar used in the medium The lowest pH attained with fructose was 5.3; glucose 3.5; sucrose, 4.0; and an equimolecular amount of fructose and glucose, 3.5. When lactose w^as used in combination with these amino acids, the pH of the culture medium remained essentially constant at 7.0 Any changes in environmental factors which affect the rate of growth of a fungus may also affect the changes in pH of the culture medium. Robbins and Schmitt (1945) found that the time required for Phycomyces blakesleeanus to lower the pH of a glucose-asparagine medium to a given level was a function of temperature of incubation. Growth and the production of acid were more rapid at 26°C than at 20°C. The rate at which the pH of a culture medium is changed by a fungus is also depend- ent upon the volume of medium used in flasks of the same size. Some of our data which illustrate this for Sordaria fimicola are shown in Fig. 27. The time required for these cultures to attain maximum weight and to produce perithecia correlated with the changes in pH. Effect of acidity on media. The composition of a medium may be changed as a result of changing the pH. The various cations and anions may combine to form insoluble compounds at certain pH values. Mag- nesium and phosphate ions are compatible in acidic solutions, but as the concentration of hydrogen ion is decreased, these ions combine to form an insoluble compound, the solubility of which becomes less as the pH is increased. Calcium phosphate is likewise less soluble in alakline solu- tions. Ferric iron may be largely removed from media as either the hydroxide or the phosphate, by making the media alkaline. If an alkaline medium is filtered, certain constituents will be removed to a greater or lesser extent. Lilly and Leonian (1945) found that by making a medium alkaline to pH 8 and filtering, the iron concentration w^as lowered to such levels that Rhizohium trifolii made about one-fifth as much growth as when 250 jug of iron per liter was added to the medium. If a precipitate is not removed by filtration, the situation is different. Any insoluble precipitate is in equilibrium with the dissolved compound, as indicated below. FeP04 ^ FeP04 ^ Fe+ + + + PO4" solid in solution ionized HYDROGEN-ION CONCENTRATION 1C5 As the ions are utilized, more and more of the precipitate will dissolve. The effect of a change in pH of the solution as a result of the metabolic activities of the fungus must be considered. An acid reaction will hasten solution of the precipitate, while an increase in alkalinity will delay the process. It is possible that the harmful effects sometimes noted in alkaline media may be due, in part, to an induced iron deficiency. The influence of pH on the solubility of certain ions may be modified by the presence of other compounds, especially those which form com- plexes. The solubility of iron in alkaline solutions is greatly increased in the presence of hydroxy organic acids such as citric, tartaric, and malic acids. Ammonia and amino acids also form complexes with certain ions, e.g., copper. The presence of any complex-forming compound may modify the availability of the ions with which it forms complexes. The chemical changes in media due to alteration of pH, whether imposed from the outside or caused by the fungus, affect metabolic processes. The pH of a culture medium changes during the growth of a fungus, and these changes may affect the composition of the medium and thus the response of the fungus. pH and oxygen supply. The solubility of oxygen in water is slight, being less than 10 mg. per liter at 20°C. The rate of diffusion of oxygen into media is dependent upon the composition and the pH. Rahn and Richardson (1941) have described a simple and elegant method of measur- ing the rate of diffusion of oxygen into agar media. Methylene blue, an organic dye which is colorless when reduced and blue when oxidized, w^as used as an indicator. When this dye (1/200,000) is autoclaved with media which contain easily oxidized constituents such as glucose, the dye is reduced to the leuco, or colorless, form. As oxygen diffuses into the medium, the reduced dye is oxidized, and the rate at which the blue zone advances into the medium is a measure of the rate of oxygen diffusion. The pH of the medium also affects the ease with which certain constitu- ents are oxidized. Some data of Rahn and Richardson (1941) on the rate of oxygen diffusion into a peptone medium are shown in Fig. 28. The amount of oxygen available to submerged mycelium is greater in acidic than in alkaline media. Effect of pH on utilization of nutrients. Before any substance (ion or molecule) is utilized, it must first pass through the cell wall and the protoplasmic membrane. The cell wall is nonliving and consists of polysaccharide-like compounds. For a discussion of the nature of the cell wall and literature citations, see Brian (1949). The protoplasmic membrane appears to be composed of proteins and lipoid-protein com- plexes. Proteins are colloidal amphoteric compounds. An amphoteric compound possesses both acidic and basic properties and may form salts with either acids or bases. The protoplasmic membrane has acidic 166 PHYSIOLOGY OF THE FUNGI properties due to carboxyl and sulfhydryl groups and basic properties by virtue of having amino and other basic groups. The protophismic mem- brane, therefore, should form salt-hke compounds with both cations and anions. Bacteria are considered by McCalla (1940) to act as ion-exchange sub- stances, and fungus spores have been shown to act in the same manner. McCalla investigated ion replacement by saturating cells of Escherichia 60 Time in hours Fig. 28. The effect of hydrogen-ion concentration on the rate of diffusion of oxygen into 1 per cent peptone in phosphate buffer. Leucomethylcne bkie was used as an indicator. The rate of penetration of oxygen with time was followed by measuring the depth of the blue zone. (Drawn from the data of Rahn and Richardson, Jour. Bad. 41 : 240, 1941. By permission of The Williams & Wilkins Company.) coli with magnesium ion and tested the replacing effects of other cations. Sodium and potassium ions replaced only a little magnesium, while hydro- gen and calcium ions were much more effective. From this viewpoint the relative amounts of the various cations adsorbed from a medium would be a function of the concentration of the ions present and the relative affinity of the membrane proteins for the different cations. The concentrations of the hydrogen and hydroxyl ions in a culture medium change during growth and may act to regulate the adsorption of other ions. The pH of the culture medium may alter the relative adsorption of other ions which are essential to nutrition or which are toxic. At the lower pH limit the protoplasmic membrane may be so thoroughly saturated with hydrogen ions that the essential cations are unable to enter the cell in adequate amounts. The same situation would HYDROGEN-ION CONCENTRATION 1G7 exist at the limiting alkaline pH values, except that it is the adsorption of essential anions which would be hindered by hydroxyl ions. A satisfactory explanation of all the phenomena involved in cell perme- ability is lacking. It is known that the external pll affects the absorption of various compounds, particularly those which ionize. The mycelium of Aspergillus niger takes up acid dyes, such as light green and methyl orange, when the external pH is 3.1 or less. Basic dyes such as methylene blue and neutral red are absorbed only when the external pH is greater than 3.1 (Biinning, 193G). These dyes escaped from the cells only when the external pH was in the same range in which these dyes were absorbed. Wyss ct al. (1944) found the utilization of p-aminobenzoic acid by a deficient mutant of Neurospora crassa to be greatly increased in acidic media. The ionization constant of p-aminobenzoic acid is about 2 X 10'"'' {pKa, about 4.8). Therefore, at pH 3.8 about 90 per cent of the metabolite would exist in the form of the free acid, and at pH 5.8 only 10 per cent would be in this form. It was found that about eight times as much of this vitamin was required at pH 6.0 as at pH 4.0 to support the same amount of growth (see Fig. 41). On theoretical grounds, it is probable that the pH of the medium would affect the utilization of other vitamins which are weak acids (biotin, pantothenic and nicotinic acids). The external pH of the medium has been shown to affect the internal pH of fungus cells. By changing the external pH and by using indica- tors, Biinning (1936) found the internal pH of Aspergillus niger cells could be changed between 4.2 and 5.0 without injuring the cells. Greater changes in internal pH were possible, but injury and death ensued. Armstrong (1929) crushed the fruit bodies of a number of fleshy fungi and measured the pH of the expressed juice. The pH range of these liquids was 5.9 to 6.2. At best these are but average values. It is well known that the external pH may affect certain processes within the fungus cells. For example, growth of Sordaria fimicola in glucose-casein hydrolysate medium was slow when the initial pH of the medium was 4.0, but when the initial pH of the medium was 3.6 to 3.8, normal development did not occur (Lilly and Barnett, 1947). This failure to grow was traced to a thiamine deficiency, for when thiamine was added to the medium (initial pH 3.6 to 3.8), normal growth and perithecial formation took place. It appears possible that the low exter- nal pH may have lowered the internal pH to such an extent that the synthesis of thiamine was prevented (this fungus is self-sufficient for thiamine when the pH of the medium is 4 or greater). These effects are shown in Figs. 38 and 40. Additional evidence indicated that these conclusions are correct, for pyruvic acid accumulated in the culture medium when the initial pH was 3.6 to 3.8. On the addition of thiamine this acid disappeared from the culture medium. 168 PHYSIOLOGY OF THE FUNGI SUMMARY All aqueous solutions contain hj^drogen and hydroxyl ions. Hydrogen- ion concentration is most often expressed in terms of S0rensen's scale of pH. The pH scale is logarithmic. Acidity and alkalinity are expressed on the same scale. A pH of 7 indicates equivalent concentrations of hydrogen and hydroxyl ions. Values of less than 7 indicate acidity, and pH values greater than 7 indicate alkalinity. The smaller the pH values, the greater the concentration of hydrogen ions. Buffers are substances which tend to maintain the pH of a solution constant when either strong acid or strong alkali is added or when the solution is diluted with water. Mixtures of weak acids or bases and their soluble salts, and amphoteric compounds such as amino acids and proteins act as buffers. The pH range over which a given buffer is effec- tive is a function of the ionization constant of the weak acid (Ka) or base (Kb) from which the buffer is made. The effective pH range of a simple buffer is two pH units. The upper and lower pH values between which a fungus is able to grow is called the pH range. The pH ranges of various species are different. Fungi generally tolerate more acid than alkali. The optimum pH range may be different for growth and sporulation. The pH of a medium in which a fungus is growing may change. High buffer concentration and limited growth may keep the changes in pH at a minimum. To follow the changes in pH of a culture medium, frequent determinations should be made. Four metabolic processes operate to change the pH of a culture medium : (1) utilization of cations, (2) utilization of anions, (3) formation of acids from neutral metabolites (especially carbohydrates), and (4) formation of bases (especially ammonia) from amino acids and proteins. The net change in pH is the result of the interaction of all of these processes, REFERENCES Armstrong, J. I.: Hydrogen-ion phenomena in plants. Hydrion concentration and buffers in the fungi, Protoplasma 8: 222-260, 1929. Brian, P. W.: Studies on the biological activity of griseofulvin, Ann. Botany (N.S.) 13:59-77, 1949. BiJNNiNG, E, : Ueber Farbstoff- und Nitrataufnahme bei Aspergillus niger, Flora 131:87-112, 1930. Canting, E. C: The physiology of the aquatic Phycomycete, Blastocladia Pring- sheimii, with emphasis on its nutrition and metabolism, Am. Jour. Botany 36: 95-112, 1949. *DiMOND, A. E., and G. L. Peltier: ControUing the pH of cultures of PenicilUum notatum through its carbon and nitrogen nutrition. Am. Jour. Botany 32 : 46-50, 1945. Dole, M.: The Glass Electrode. Methods, Applications, and Theory, John Wiley & Sons, Inc., New York, 1941. HYDRUUEN-ION CONCENTRATION 169 ^Emerson, R., and E. C. Canting: The isolation, growth, and metabohsm of Blasto- cladia in pure culture. Am. Jour. Botany 35: 157-171, 1948. Foster, J. W.: Chemical Activities of Fungi, Academic Press, Inc., New York, 1949. GoRTNER, R. A.: Outhnes of Biochemistry, 3d ed., John Wiley & Sons, Inc., New- York, 1949. Haldane, J. B. S.r Enzymes, Longmans, Roberts and Green, London, 1930. Henry, B. W., and A. L. Andersen: Sporulation by Piricularia oryzae, Phytopathol- ogy 38: 265-278, 1948. Johnson, H. W.: Some relationships between hydrogen ion, hydroxyl ion and salt concentration and the growth of seven soil molds, loiva State Coll. Ayr. Mech, Arts Research Bull. 76, 1923. KoLTHOFF, I. M., and C. Rosenblum: Acid-base Indicators, The Macmillan Com- pany, New York, 1937. *LiLLY, V. G., and 11. L. Barxett: The influence of pH and certain growth factors on mycelial growth and perithecial formation by Sordaria fimicola, Am. Jour. Botany 34: 131-138, 1947. Lilly, V G., and L. LI. Leonian: The interrelationship of iron and certain accessory factors in the growth of Rhizobium trifolii strain 205, Jour. Bad. 50: 383-395, 1945. Lindeberg, G.: Ueber die Physiologie Ligninabbauender Bodenhymenomyzeten, Symbolae Botan. Upsalienses 8(2): 1-183, 1944. LiNGANE, J. J. : Saturated potassium hydrogen tartrate solutions as a pH standard. Anal. Chem. 19: 810-811, 1947. LuNDEGARDH, H. : Dcr Einfluss der WasserstofRonenkonzentration in Gegenwart von Salzen auf das Wachstum von Gibherella Saubinetti, Biochem. Zeit. 146: 564-572, 1924. McCalla, T. M.: Cation adsorption by bacteria. Jour. Bad. 40: 23-32, 1940. Mathur, R. S., II. L. Barnett, and V. G. Lilly: Sporulation of Colletotrichum Imdemuthianum in cultvire. Phytopathology 40: 104-114, 1950. Meacham, M. R.: Note upon the hydrogen ion concentration necessary to inhibit the growth of four wood-destroying fungi, Science 48: 499-500, 1918. Pehrson, S. O.: Studies on the growth physiology of Phacidium infestans Karst., Physiologia Plantar um 1: 38-56, 1948. Pritham, G. H., and A. K. Anderson: The carbon metabolism of Fusarium lyco- persici on glucose, Jour. Agr. Research 55 : 937-949, 1937. Rahn, O., and G. L. Richardson: Oxygen demand and oxygen supply. Jour. Bad. 41: 225-249, 1941. RoBBixs, W. J.: Isoelectric points for the mycelium of fungi, Jour. Gen. Physiol. 6: 259-271, 1924. *RoBBiNS, W. J., and M. B. Schmitt: Factor Z2 and gametic reproduction by Phy- comyces, Am. Jour. Botany 32 : 320-326, 1945. Ryan, J. F., G. W. Beadle, and E. L. Tatum: The tube method of measuring the growth rate of Neurospora, Am. Jour. Botany 30 : 784-799, 1943. *Scott, I. T.: The influence of hydrogen-ion concentration on the growth of Fusarium lycopersici and on tomato wilt, Missouri Coll. Agr. Research Bull. 64, 1924. Snell, F. D., and C. T. Snell: Colorimetric INIethods of Analysis, 3d ed., D. Van Nostrand Company, Inc., New York, 1948. Starkey, R. L., and S. A. Waksman: Fuiigi tolerant to extreme acidity and high concentrations of copper sulfate, Jour. Bud. 4C : 509-519, 1943. Tamiya, H.: Studi^n liber die Stoffwechseiphysiologie von Aspergillus oryzae. II. Acta Phyiochim. {Japan) 4: 77-213, 1928. Umbreit, W. W., R. IL Burris, and -J. F. Staufj^es? Manomptrjc Techniques and 170 PHYSIOLOGY OF THE FUNGI Related Methods for the Study of Tissue MetaboUsm, Burgess Publishing Co, Minneapolis, 1945. , , . , . .1 j Van Slyke, D. D.: The kinetics of hydrolytic enzymes and their bearmg on methods of measuring enzyme activity, Advances in Enzymol. 2 : 33-47, 1942. WoLPERT F. S.: Studies in the physiology of the fungi. XVII. The growth of certain wood-destroying fungi in relation to the H-ion concentration of the media, Ann. Missouri Botan. Garden 11: 43-97, 1924. Wyss, O., V. G. Lilly, and L. H. Leonian: The effect of pH on the availability of p-am'inobenzoic acid to Neurospora crassa, Science, 99 : 18-19, 1944. CHAPTER 9 VITAMINS AND GROWTH FACTORS It is kno^\^l that, for normal growth and development, animals and man require in their diet minute amounts of certain organic compounds, in addition to those which yield energy or are used for structural purposes. Similarly, certain fungi must obtain from the substrate some of the same substances for growth, reproduction, and other vital functions. Other fungi are able to synthesize these compounds, which are called groivth factors, or vitamins. Both terms have often been applied to the same compounds, although the terms are not always synonymous. Originally, the term vitamin was applied to the accessory factors in animal nutrition, and some workers would restrict its use to animals and man. The term growth factor has a somewhat broader connotation than vitamin. It includes the components and derivatives of some vitamins, as well as other compounds which cannot be classified otherwise at present. The chemical names of the vitamins also may be used. GENERAL CONSIDERATIONS A number of vitamins, such as thiamine and biotin, have been shown to perform definite functions in fungi as well as in animals, and there is no reason to assume that the fundamental functions in the two groups of organisms are essentially different. The characteristic features of a growth factor (vitamin) include the following: (1) its organic nature; (2) its activity in minute quantities; (3) its catalytic action; (4) the specificity of its action. It is known that some vitamins are components of enzyme systems, and it may be assumed that all act in this way. In the fungi the relative effects of the presence of vitamins in the medium usually are measured by the resultant vegetative growth, although vitamins are known to affect reproduction and other processes. Needless to say, studies of vitamin deficiencies must be carried out under carefully controlled conditions, using clean glassware, purified chemicals, and precaution against contamination. Despite all precautions possible, variable results often occur, and tests may need to be repeated several times before the vitamin deficiencies of some fungi can be definitely determined. SYNTHESIS OF VITAMINS BY FUNGI Many fungi are able to grow and develop normally on a substrate con- taining no vitamins. For example, Aspergilhis niger grows well on a 171 172 PHYSIOLOGY OF THE FUNGI synthetic medium composed of pure chemicals (glucose, asparagine, salts, and micro elements). Phycomyces hlakesleeaniis makes no growth on this medium unless thiamine is added. We may conclude that A. niger either does not need thiamine in its metabolism or is capable of synthesiz- ing from the compounds of the medium all vitamins in sufficient quantities to meet its needs. The growth of P. blakesleeanus on the culture filtrate of A. niger is proof that thiamine is synthesized by the latter species. Thus, A. niger may be called a self-sufficient fungus with respect to vitamins. Schopfer (1943) has applied the term autotrophic with respect to vitamins to this group of organisms. The detection of self-sufficient fungi in the laboratory is dependent upon their ability to grow on vitamin- free synthetic media containing suitable sources of carbon and nitrogen. A discussion of the economic importance of certain vitamins as metabolic products of fungi is given in Chap. 13. Some fungi which have been reported to be self-sufficient with respect to vitamins are listed below: Aspergillus (most species tested) Basisporium gallarum Botrytis allii Cercospora apii C. beticola Chaetomium globosum Cordyceps militaris Daldinia concentrica Fusarium (most species tested) Glomerella cingulata Helminthosporium gramineum H. victoriae Monascus purpurea Monilinia fructicola (some isolates) Neocosmopara vasinfecta Penicillium (most species tested) Phoma betae Rhizopus nigricans Sclerotinia sclerotiorum Septoria nodorum Sphaeropsis malorum Ustilago striiformis Growth curves of Chaetomium globosum are presented in Fig. 29, as an example of a self-sufficient fungus. It is evident that good mycelial growth was made in the vitamin-free medium and that the addition of four vitamins caused no significant increase in the rate of growth at any time. VITAMIN DEFICIENCIES IN FUNGI As pointed out above, some fungi do not grow on synthetic media composed of pure chemicals, because they are unable to synthesize certain vitamins. These fungi have been called variously vitamin-deficient, vitaminless, or heterotrophic with respect to one or more specific vitamins. We prefer to use the term vitamin-deficient, following Robbins and Kavanagh (1942). Vitamin deficiencies among the fungi have been detected only for certain members of the water-soluble B-complex group. The most common vitamins involved are thiamine, biotin, inositol, pyridoxine, nicotinic acid, and pantothenic acid. Vitamin deficiencies can be detected accurately only on synthetic media which, other than VITAMINS 173 E O -^^■"^^ ^ ^^^— — 1 250 •'^ ^"^ y^'* 4 vitamins— _^ present >^ ^ 200 A r/^ ^^ 150 > / (/ // 1 //" 1 ~ Without vitamins 100 // // > / 50 // // n ^ ^ 10 12 14 0 2 4 6 8 Days of incubation Fig. 29. Growth curves of Chaetomiurn globosum, a self-sufficient fungus, in 25 ml. of liquid glucose-casein hydrolysate medium in the absence of vitamins and when thiamine, biotin, inositol, and pyridoxine were added. Fig. 30. Mutualistic symbiosis with regard to vitamins. Phycomyces blakesleeanus, thiamine-deficient, inoculated on the right and Sordaria fimicola, biotin-deficient, inoculated on the left. Both fungi made only slight growth until the two colonies met. Note the perithecia of Sordaria on the right and the sporangiophores of Phycomyces on the left. Each fungus excreted into the medium the vitamin which the other could not synthesize. 174 I'HYSIOLOGY OF THE FUNGI vitamins, meet all the requirements for normal growth and development of the fungus under study. The effect of one deficient fungus on another is shown in Fig. 30. Methods of detecting vitamin deficiencies. Tests for vitamin deficiencies of fungi are not difficult to perform, but they do require clean glassware and careful preparation of media. It is convenient to conduct pre- liminary experiments using only the four vitamins (thiamine, biotin, inositol, pyridoxine) for which fungi are most frequently deficient. A greater number of vitamins may be included in subsequent tests if a fungus does not grow well on any of the media first used. Either agar or liquid media may be used, and the visual measure of growth is satis- factory for the screening tests. A simple and convenient method for preliminary tests for deficiencies in filamentous fungi isolated from nature is by the use of agar media in test tubes, as shown in Fig. 31. Slight growth on agar media without added vitamins may be due to impurities in the medium. A high percentage of the deficiencies will be detected by this method, since deficiencies for only one or two vitamins are com- mon among the filamentous fungi. After the deficiencies have been identified by preliminary experiments, it is then highly desirable to grow a fungus in liquid media, so that the mycelium may be harvested and dry weights determined (see Suggested Laboratory Exercises for direc- tions). The casein hydrolysate-glucose medium, given in Chap. 10, has proved quite satisfactory for accurate vitamin studies. From the dry weights of cultures determined at intervals throughout the growth period of a fungus, growth curves may be plotted. Such curves are necessary for accurate interpretations of the effects of vitamins in the medium. A somewhat different method is used by Burkholder (1943) for defi- ciency studies of yeasts, where deficiencies for more than two vitamins are common. This method is illustrated in Fig. 32. A deficiency is detected by the inability to grow in a medium which is complete except for one vitamin. Failure to grow in a medium indicates a deficiency for the vitamin omitted. Liquid media in test tubes are used for yeasts, so that growth may be measured by photoelectric colorimeter. Total and partial deficiencies. Phycomyces hlakesleeanus was widely used in the early studies of thiamine. Schopfer established the deficiency for thiamine and determined the requirements for this vitamin. Schopfer's graph (Fig. 33) shows the growth curves of the fungus over a period when different amounts of thiamine were added to the basal culture medium (Schopfer, 1943). The fact that no growth occurred in the medium lacking thiamine is not shown by the graph. An increase in both the rate and the total amount of growth, as the amount of thiamine is increased, is clearly shown between the fifth and seventh days. Thus P. hlakeslseanus is unable to synthesize thiamine, which it must obtain VITAMINS 175 A B C D E Fig. 31. Method of detecting common vitamin deficiencies of filamentous fungi. Deficiencies are evident by failure to grow on media lacking the necessary vitamin or vitamins. The above media contained: A, no vitamins; B, thiamine; C, bio tin; D, thiamine and biotin; E, thiamine, biotin, inositol, and pyridoxine. The fungi, from top to bottom, are Ceratostomella fimbriata, Sordaria fimicola, Pleurage curvicolla, and C. ulmi. 1 176 PHYSIOLOGY OF THE FUNGI Fig. 32. Method of detecting multiple vitamin deficiencies of yeasts. Failure to grow in the absence of a particular vitamin indicates a deficiency if the culture grew in a medium supplied with a combination of vitamins. Growth of a strain of Saccharo- myces cerevisiae (above) and Mycoderma valida (below) after 5 days at 25°C. From left to right the vitamin supplements were: Tube 1, none; 2, less thiamine; 3, less pantothenic acid; 4, less pyridoxine; 5," less inositol; 6, less bio tin; 7, less nicotinic acid ; 8, all six vitamins. VITAMINS 177 from its substrate. Figure 33 emphasizes two important features which must be considered in vitamin studies: (1) the effects of different amounts of the vitamin in the medium, and (2) the response of the fungus over a period of time sufficiently long to allow maximum growth. The three- dimensional graph permits one to plot dry weight against both variables. The failure of a fungus to make an appreciable amount of growth even after an extended period of incubation on a medium essentially free of a Asparagine 0.1 % r, 9 ^ Mg. ^90 Fig. 33. Three-dimensional graph showing growth of Phycomyces blakesleeanus on a, synthetic medium as a function of thiamine concentration and time. (Courtesy of Schopfer, Protoplasma 28: 383, 1937; also from the book "Plants and Vitamins," p. 102, 1943. PubUshed by permission of Chronica Botanica Co.) particular vitamin, like the case illustrated by P. hlakesleeanus and thiamine, indicates that the deficiency is total; i.e., the synthesis of that vitamin is zero. Vitamin deficiencies of many fungi are only partial, as shown by a slower rate of growth in a vitamin-free medium than in the presence of added vitamins. The degree of partial deficiency may vary M^dely, from slight to nearly total. Partial deficiencies may be easily overlooked by terminating an experiment too soon. An incubation period of 1 or 2 months is often required to distinguish between partial and total deficiencies of some fungi. An example of partial thiamine deficiency is illustrated by Lenzites trabea (Fig. 34). In a medium containing thiamine, maximum Aveight 178 PHYSIOLOGY OF THE FUNGI was attained in 20 days, while, in medium lacking thiamine, the fungus required approximately 40 days to reach the maximum weight. This is attributed to the slow rate of synthesis of thiamine. Other isolates of L. trahea showed varying degrees of partial deficiency (Lilly and Barnett, 1948). Single and multiple deficiencies. The above discussion has dealt with examples of single deficiencies (for a single vitamin). For example, Sordaria fimicola is deficient only for biotin (Fig. 31), Lenzites trahea. EO 30 40 Incubation (days) Fig. 34. Growth of a haploid isolate of Lenzites trahea and change in pH of liquid glucose-casein hydrolysate medium at 25°C., with and witliout the addition of thiamine. These curves illustrate a partial deficiency for thiamine. (After Lilly and Barnett, Jour. Agr. Research 77: 290, 1948.) Ceratostomella Hmhriata (Fig. 31), and Phycomyces blakesleeanus for thiamine only. On the other hand, some fungi have multiple deficiencies (for two or more vitamins). These may be total or partial. An illustra- tion of multiple deficiency is furnished by Sclerotinia camelliae (Fig. 35). Little or no growth occurred on vitamin-free medium or that containing either thiamine or biotin alone; the fungus grew well only in media con- taining both thiamine and biotin. When inositol also was added, growth was consistently better than in the presence of the two vitamins. This indicates a partial deficiency for inositol, in addition to the total, or near total, deficiencies for thiamine and biotin. Pyridoxine, when added to the other three vitamins, had little or no effect on growth under these conditions. Other examples of multiple vitamin deficiencies are common. Pleurage curvicolla (Fig. 31), Chaetomium convolutum, Coemansia interrupta, and VITAMINS 179 400 10 12 14 16 Days of incubation Fig. 35. Growth of Sclerotinia camelliae in 25 ml. of liquid glucose-casein hydrolysate medium at 25°C. Note the nearly total deficiency for biotin and the partial deficiency for inositol. Failure to grow in thiamine alone and in the absence of vitamins indicates total deficiency for thiamine. 6 12 18 24 Days of incubation Fig. 36. Growth of Lambertella pruni in 25 ml. of liquid glucose-casein hydrolysate medium containing various vitamins. Partial deficiencies for both thiamine and biotin are evident, being greater for thiamine. Note that the addition of inositol and pyridoxine to media containing thiamine and biotin depressed growth. 180 PHYSIOLOGY OF THE FUNGI Ophiobolus graminis are highly or totally deficient for both thiamine and biotin. Partial deficiencies for both thiamine and biotin are illustrated by Lambertella pruni (Fig. 36). Slight growth in the control and excellent growth only in media containing both thiamine and biotin identify the deficiencies. Intermediate growth in thiamine alone and in biotin alone shows that the deficiencies are partial. The synthetic capacity is rela- tively greater for biotin than for thiamine. The deficiencies of Endothia parasitica are similar to those of L. pruni. Blastodadia pringsheimii was 30 -^ 20 E o> E ^^ 4* .5* i A 10 » ^* » • Biotin • < Thiamin weight (mgms) D o c ♦' i ^^* ■ — 1 y^ Nicotinamide / 1 .. 1 1 Q — 1 — 1 — t 1 • 1 • i_ 05 1.0 1.5 Micrograms per 75 cc. ' 1 1 1 1 2.0 0 0.002 0.015 0.005 0.01 Micrograms per 75 cc. Fig. 37. The effect of concentration of essential vitamins on dry weight of Blasto- dadia pringsheimii. (Courtesy of Cantino, Am. Jour. Botany 35: 241, 1948.) reported (Cantino, 1948) to be partially deficient for thiamine and biotin and nearly totally deficient for nicotinic acid (Fig. 37). Cerotostomella ips No. 255 was shown to be completely deficient for thiamine, biotin, and pyridoxine (Ptobbins and Ma, 1942a). Multiple vitamin deficiencies are more common among the yeasts than among the filamentous fungi, and some yeasts show deficiencies not known to exist in filamentous fungi isolated from nature. For these reasons the yeasts as a group have received much attention in vitamin investigations. The vitamin requirements of 38 species and strains of yeast were reported by Burkholder (1943), and for 110 additional named species and varieties by Burkholder et at. (1944). A summary of the deficiencies reported in these two papers is as follows: biotin, 114; thia- mine, 48; pantothenic acid, 44; inositol, 19; nicotinic acid, 19; pyridoxine, 19. No deficiency for riboflavin was found. Several isolates were deficient for three or more vitamins. Saccharomyces oviformis was deficient for biotin, pantothenic acid, and pyridoxine, while S. mace- doniensis Y-91 showed complete or partial deficiencies for thiamine, VITAMINS 181 pantothenic acid, nicotinic acid, and biotin. S. ludwigii Y-974 and Kloeckera brevis were totally or partially deficient for six vitamins (thia- mine, biotin, inositol, pyridoxine, nicotinic acid, and pantothenic acid). Growth of all of the 38 yeasts reported by Burkholder (1943) was increased by the addition of liver extract to the medium containing the seven vitamins. The preceding discussion of the effects of added vitamins on the growth of fungi has been based on the assumption that near-optimum amounts of the vitamins were present in the media. However, the optimum 20 28 Days of incubation Fig. 38. The effect of concentration of biotin on the rate and amount of growth ot Sordaria fimicola in 25 ml. liquid glucose-casein hydrolysate medium, initial pH 4.4. Growth in this medium containing biotin but no thiamine is evidence that this fungus can synthesize thiamine under these conditions. (After Lilly and Barnett, Am. Jour. Botany 34: 134, 1947.) amount of a vitamin may vary with changes in other conditions and may be different for different fungi. We have found that the following amounts per liter of the four commonly needed vitamins are near optimum for many filamentous fungi: thiamine, 100 Mg; pyridoxine, 100 jug; biotin, 5 /ig; inositol, 5 mg. The effects of biotin concentration on the growth of Sordaria fimicola are illustrated in Fig. 38, which shows a decided increase in growth rate with greater amounts of biotin. Growth was most rapid in a medium containing 6.4 jug biotin per liter (0.16 /zg per flask), but a steady slow increase in dry weight is evident in as low as 0.1 ^g biotin per Hter. Absolute and conditioned deficiencies. According to Robbins and Kavanagh (1942), the deficiency of a fungus for a specific vitamin may be absolute or conditioned. Phycomyces blakesleeanus and Ceratostomella 182 PHYSIOLOGY OF THE FUNGI fimbriata show absolute total deficiencies for thiamine. No environ- mental condition is known to allow the synthesis of this vitamin by these fungi. In the case of a conditioned deficiency, the synthesis of the vitamin may be influenced by certain environmental conditions, such as temperature, composition, concentration, and pH of the medium. Pythium hutleri failed to grow in a mineral salts-asparagine medium containing 16.4 g. of salts per liter unless thiamine was added (Robbins and Kavanagh, 1938). When the salt concentration was reduced to 1.64 g. per liter, this species grew without the addition of thiamine. A defi- 30 35 40 45 Temperature Fig. 39. Growth-temperature relations for wild-type Neurospora and a temperature- sensitive mutant deficient for riboflavin. Amounts of riboflavin are indicated on the curves in micrograms per 20 ml. of medium. Below 25°C. growth was good without riboflavin, while no growth occurred above 28°C. without added riboflavin. (Courtesy of Mitchell and Houlahan. Am. Jour. Botany 33: 31, 1946.) ciency for riboflavin conditioned by temperature was reported by Mitchell and Houlahan (1946) for a mutant of Neurospora (Fig. 39). Growth was poor or none at temperatures above 25°C. unless riboflavin was added. Below 25°C. the fungus was able to synthesize riboflavin. The partial deficiency of Sclerotinia camelUae for inositol was influenced by tempera- ture, particularly in the above-optimum range (Barnett and Lilly, 1948). Low pH of the medium resulted in partial thiamine deficiency of Sordaria fimicola, while no deficiency for thiamine was apparent at initial pH 4.0 or above (Lilly and Barnett, 1947). Within the range of 3.8 to 3.4, growth was quite slow, but the addition of thiamine overcame the inhibition due to the high acidity (Fig. 40). These results indicate that pH 3.8 or lower inhibits the synthesis of thiamine by S. fimicola. In a similar way, the availability of p-aminobenzoic acid to a mutant of VITAMINS 183 10 14 Days of incubation Fig. 40. Effect of concentration of thiamine on the rate and amount of growth of Sordaria fiynicola in 25 ml. Hquid glucose-casein hydrolysate medium, initial pH 3.8. Compare with Fig. 38. (After Lilly and Barnett, Am. Jour. Botany 34: 134, 1947.) 40 30 T5 O20 10 PH4.0 / ^ < / pHj^ — •■ :— — T * H ^ .^ f ( ,^ pH 6.0 , -< ^ / / y // / / / pHTCL ^ z' / C^ i — 0.05 0.1 0.2 0.3 0.4 Microgroms p-aminobenzoic ocid per 25 ml. medium 0.5 Fig. 41. Effect of concentration of p-aminobenzoic acid at different pH values on the growth of a mutant of N eurosj}ora crassa deficient for this vitamin. The fungus was grown on liquid glucose-casein hydrolysate medium for 3 days. (Drawn from the data of Wyss, Lilly, and Leonian, Science 99: 18, 1944.) 184 PHYSIOLOGY OF THE FUNGJ Neurospora crassa deficient for this vitamin was found to be influenced by the pH of the medium (Wyss et al, 1944) (Fig. 41). The abiHty of a mutant of Neurospora sitophila to synthesize pyridoxine was shown to be dependent not only on the pH of the medium but also on the source of nitrogen (Stokes et al., 1943). When nitrate, amino, amide, or certain other nitrogen compounds served as the nitrogen source, no growth occurred without the addition of pyridoxine. However, in the presence of ammonium salts, growth occurred at an initial pH range of 5.6 to 7.3, without added pyridoxine. In this pH range, free ammonia is formed. In the absence of free ammonia, the pyridoxine synthesized is unavailable to this mutant (Strauss, 1951). According to Fromageot and Tschang (1938), the red yeast, Rhodotorula sanniei, requires thiamine when the carbon source is glucose, but when redistilled glycerol replaces glucose, thiamine is not needed. It is inter- esting to speculate whether this fungus is better able to synthesize thia- mine in a glycerol medium or whether much less thiamine is required to metabolize glycerol than glucose. The concentration of the micro essential elements has also been shown to influence the synthesis of vitamins by microorganisms (see Chap. 13 for specific information). INHIBITORY EFFECTS OF VITAMINS In certain cases vitamins may have an inhibitory effect on growth, particularly when present in excessive dosages. The interrelated effects of temperature and amount of inositol were described (Barnett and Lilly, 1948) for Sclerotinia camelliae. At a temperature below 26°C. the partial deficiency was overcome by adding 5 mg. inositol to the medium. Above 26°C. the fungus was highly sensitive to small changes in temperature and in amounts of inositol in the medium (Fig. 42). The same amount of inositol which stimulated grow^th at or below 26°C. was strongly inhibi- tory at 27°C. Increased amounts of inositol caused greater inhibition of growth. Since the maximum temperature for growth is slightly above 27°C., it is believed that, as the temperature approaches this point, the fungus becomes highly sensitive to the increased amounts of inositol in the medium. Some vitamins are known to have a depressing effect on growth of certain fungi not deficient for these particular vitamins. For example, Fig. 36 shows that Lambertella pruni produces more dry weight in the presence of both thiamine and biotin than when inositol and pyridoxine are also added to the medium. Similarly, it is reported (Elliott, 1949) that, for a self-sufficient isolate of Fusarium avenaceum, both the rate of growth and maximum amount of mycelium were greater in vitamin-free medium than when vitamins were added. The presence of thiamine also VITAMINS 185 Fig. 42. The interrelated inhibitory effects of high concentrations of inositol and near-maximum temperatures on the growth of Sclerotinia camdliae, which is partially deficient for inositol below 26°C. Cultures 19 days old. Thiamine and biotin were added to all media. Temperatures ±0.3°C. Left to right: 26°C., 26.6°C., 27°C. Amounts of inositol added per liter were: A, none; B, 1 mg.; C, 10 mg.; D, 100 mg. 186 PHYSIOLOGY OF THE FUNGI has been reported to depress the growth of several fungi, including Collctotrichum lindemuthianum (Mathur et al., 1950), Rhizopus suinus (fechopfer and Guilloud, 1945), and Fusarium lini (Wirth and Nord, 1942). Other cases have been observed in our laboratory. In the case of Rhizopus suinus, the addition of inositol overcame the inhibitory effects of thiamine, and we believe it to be effective with certain other fungi. On the basis of these reports, it would seem unwise to add vitamins indiscriminately to media used for the study of fungi which are self- sufficient for these vitamins. VITAMERS Certain microorganisms are less specific in their vitamin requirements than are animals, owing apparently to their greater synthetic ability. Some vitamin-deficient fungi may respond well to one of the vitamin moieties, as in the case of thiamine, or to a compound similar to the vitamin. The term vitamer w^as suggested by Burk et al. (1944) to denote a compound having vitamin activity but differing in molecular structure from the true vitamin. Usually the structure of a vitamer is closely related to that of the vitamin. More specifically, these compounds are known as thiamine vitamers, biotin vitamers, etc. In general, a vitamer is active for fewer fungi than is the vitamin. Some vitamers are anti- vitamins. This topic is discussed in Chap. 11. UNIDENTIFIED GROWTH FACTORS It is quite probable that some fungi wdll be discovered which are defi- cient for vitamins or other growth factors w^hich are at present unknown. Fungi which fail to grow in synthetic media to which all the known growth factors have been added offer a challenge and an opportunity to the investigator. Burkholder and Moyer (1943) reported that Candida albicans 475 and Mycoderma vini 939 did not grow unless liver extract was added to glucose-asparagine medium containing six vitamins. One may speculate that the effect of liver extract was due to some amino acid or to an undetermined growth factor, possibly vitamin B12, which is known to be present in liver. In view of the common experience regard- ing the stimulating effect of natural substances on growth of fungi, it is evident that much more investigation on this phase of nutrition is needed. SPECIFIC VITAMINS In the first portion of this chapter the general aspects of vitamins and growth factors were considered. Different types of vitamin deficiencies and the methods of detecting deficiencies were discussed. The second portion deals with the specific vitamins, their characteristics and functions. VITAMINS 187 THIAMINE AND ITS MOIETIES Thiamine (vitamin Bi, aneurine) was the first vitamin shown to be required by a filamentous fungus. Thiamine deficiency in man is known as beriberi. Certain fungi and other microorganisms resemble man in that they are unable to synthesize this vitamin. It is probably required in the metabolism of all forms of life, and its function, to a large extent, is believed to be the same in all organisms. Schopfer (1934) demonstrated that Phycomyces blakesleeanus failed to grow in a synthetic medium unless thiamine was added. This was a stimulus for numerous studies on vitamin deficiencies of fungi. The chemical synthesis of thiamine, in 1936, was another important step in vitamin research. The student is referred to Williams and Spies (1938), Rosenberg (1942), and Schopfer (1943) for information on the history, synthesis, and natural occurrence of thiamine. The structural formula for thiamine is N=C— NHrHCl CH,— C C -CH,— N / CHs c==c— CH2— CH,OH Cl CH — S N— CH Thiamine chloride hydrochloride The thiamine molecule contains two ring structures, a substituted pyrimidine and a substituted thiazole. The pyrimidine moiety has the following formula: N====C— NH2 CH, N C— CH2X CH Thiamine pyrimidine 2-Methyl-4-amino-5-methylpyrimidine X in the substituted methyl group on C5 may be hydroxyl, chlorine, bromine, etc. The thiazole moiety has the following formula: CH3 C= =C— CH2— CH2OH N / V X CH- Thiamine thiazole 4-Methyl-5-/3-hydroxyethylthiazole 188 PHYSIOLOGY OF THE FUNGI These moieties are referred to in the Hterature as the thiamine pyrimidine and thiamine thiazole, respectively. Thiamine is somewhat unstable when exposed to alkali and heat, but at pH 3.5 it is unaffected by autoclaving. Sulfur dioxide and sulfites are destructive at pH 5 to 6. These factors must be taken into considera- tion, and it is sometimes desirable to sterilize thiamine separately, either by filtration or by autoclaving in an acidified solution. For most investi- gations, however, it is permissible to autoclave thiamine with the medium. For most fungi 100 fxg of thiamine per liter of medium is near optimum for growth and sporulation. However, the optimum varies with the amount of sugar in the medium and with other conditions. Soon after pure thiamine became available, it was discovered that certain treatments destroyed its activity for animals but did not greatly affect the potency when certain fungi were used as test organisms. The solution to this problem was reached when it became known that thia- mine, when autoclaved in the presence of alkali, was broken down into thiamine pyrimidine and thiamine thiazole. Thiamine-deficient fungi differ in their ability to utilize or synthesize the moieties of thiamine. These fungi may be classified into four groups on this basis: (1) The intact molecule of thiamine is required by some fungi which are unable to synthesize either moiety or to complete the synthesis of thiamine, even when both moieties are supplied. Examples of the group are species of Phytophthora. (2) Some other fungi, such as Phycomyces hlakesleeanus, are capable of utilizing thiamine, or of syn- thesizing thiamine when furnished with a mixture of the two thiamine moieties. (3) The addition of thiamine or thiamine pyrimidine satisfies the need of those fungi which are able to synthesize the thiazole moiety and combine it with the pyrimidine moiety to make thiamine. Examples are Parasitella simplex and Rhodotorula rubra. (4) Other fungi are able to synthesize only the thiamine pyrimidine and complete the synthesis of thiamine when furnished with the thiazole moiety. Mucor raman- nianus and Stereum frustulosum are examples. In the above discussion it was assumed that in every case the intact molecule was the active product and that neither moiety nor the presence of the two had any activity until thiamine was synthesized. Leonian and Lilly (1940) found this hypothesis to be correct. The following fungi Avere grown in a basal medium to which had been added the minimal growth factor: Fusarium niveum (none), Pythiomorpha gonapodyoides (pyrimidine), Mucor ramannianus (thiazole), Phycomyces hlakesleeanus (both moieties of thiamine), and Phytophthora erythroseptica (thiamine). After growth, the mycelium and the medium were tested for thiamine and its moieties by growing fungi of known thiamine or thiamine-moiety requirements upon media containing the mycelium extract and the medium. Some of these data are collected in Table 33. VITAMINS 189 Table 33. Assay for Thiamine and Thiamine Moieties in Mycelium and Medium Extracts of Some Fungi after Growth on Media Containing THE Minimum Growth-factor Requirements Numbers refer to relative growth on the scale of 10. (Leonian and Lilly, Plant Physiol. 15, 1940.) Test fungi and substance tested for Fungi tested and minimum vitamin requirements Pythium ascophallon (thiamine) Phycomyces blakesleeanus (both moieties) Pythiomorpha gonapodyoides (pyrimidine) Mucor ramannianus (thiazole) Fusarium niveum (none) Pythiomorpha gonapody- oides (pyrimidine) Mucor ramannianus (thiazole) Phycomyces blakesleeanus (both moieties) Phytophthora erythroseptica (thiamine) 10* 0* 10 1 1 0 10 0 10 1 10 2 10 4 3 2 10 8 8 6 10 4 10 10 5 3 10 8 10 6 10 10 8 8 8 7 10 10 10 10 * Upper figures refer to extract of mycelium, lower figures to extract of medium. It is evident that Fusarium niveum was able to synthesize thiamine from the basal medium because two test fungi which require thiamine per se grew on extracts prepared from the hyphae. The same type of proof shows that Pythiomorpha gonapodyoides synthesized thiamine when thiamine pyrimidine was added to the basal medium. Mucor raman- nianus synthesized thiamine when thiamine thiazole was added, and Phycomyces blakesleeanus synthesized thiamine when both moieties were added. In all cases the greater portion of thiamine was stored within the mycelium, and only small amounts were present in the medium. The medium extract from three fungi contained no thiamine, although appre- ciable quantities of the pyrimidine and thiazole moieties were present in all media. This shows that Phytophthora erythroseptica, for example, had broken down the thiamine molecule into its moieties, which diffused into the medium and were later utilized by certain fungi, such as Phyco- myces blakesleeanus. This suggests that in the process of its utilization thiamine is slowly destroyed. The moieties may be recombined by certain organisms but not by those which require the entire thiamine 190 PHYSIOLOGY OF THE FUNGI molecule. Robbins and Kavanagh (1941) showed that P. blakesleeanus destroyed the thiazole more rapidly than it did the pyrimidine moiety. Thus, an excess of thiazole in the mixture of the two moieties was more effective than equal quantities. They termed this the thiazole effect. Some thiamine -deficient fungi. A deficiency for thiamine is by far the most common vitamin deficiency among filamentous fungi isolated from nature. Fries (1948) states than over 200 fungi are known to be partially or totally deficient for thiamine. No doubt this is a modest estimate. Deficiency for this vitamin is more common among certain groups of fungi than others. For example, all species of Phytophthora studied have been found to require the intact molecule of thiamine. Only a few species of the true Basidiomycetes have been reported to be self-sufficient for thiamine. Many of these fungi show only partial deficiencies, while some are totally deficient. In most cases, however, there seems to be little or no correlation between thiamine deficiency and taxonomic relationship. Some common filamentous fungi (other than Basidiomycetes) which have been reported to be totally or partially deficient for thiamine or its moieties, with other deficiencies (if any) indicated in parentheses, are as follows : Blakeslea trispora, Ceratostomella fimbriata, C. ips (biotin and pyridoxine), C. montium (biotin and pyridoxine), C. pini (biotin), Chae- tomium convolutum (biotin), Choanephora cucurbitarum, Coemansia inter- rupta (biotin), Dendrophoma obscurans, Endothia parasitica (biotin), Hypoxylon pruinatum (biotin), Lambertella pruni (biotin), Lophodermium pinastri (biotin and inositol), Melanconium betulinum (biotin and inositol), Melanospora destruens (biotin), Mucor ramaymianus, Nectria coccinia, Ophiobolus graminis (biotin), Phycomyces blakesleeanus, Phytophthora spp., Piricularia oryzae (biotin), Pleurage curvicolla (biotin), Podospora curvida (biotin), Pythiomorpha gonapody aides, Pythium arrhenomanes , P. ascophallon, P. butleri, P. oligandrum, Sclerotinia camelliae (biotin and inositol), S. minor, Sordaria fimicola, certain isolates only (biotin), Thielaviopsis basicola, Valsa pini (biotin and inositol), and Xylaria hypoxylon. Reports of deficiencies for most of the above-named fungi may be found in the references for this chapter. Some few of these fungi have been studied in our laboratory and have not been previously reported as being deficient for thiamine. For thiamine-deficient yeasts see the reports of Burkholder (1943) and Burkholder et al. (1944). Mode of action. One of the primary uses of thiamine in plants and animals is for the regulation of carbohydrate metabolism. It is also probable that thiamine may be involved in other processes. A vitamin which constitutes a part of an enzyme system is known as a coenzyme. Generally a vitamin must be combined with organic or inorganic com- VITAMINS 191 pounds (or both) before it combines with the protein portion (apoenzyme) of the enzyme system. The pyrophosphoric ester of thiamine is known as cocarboxylase, or as thiamine pyrophosphate. This compound is the coenzyme of carboxylase. CH, O O N=C— NH2 I II II I C=C— CH2— CH2— O— P~0— P— OH CH3— C C— CH2— N o H H i CI CH— S N— CH Thiamine pyrophosphate (cocarboxylase) This substance is as active as thiamine (mole for mole) . Lilly and Leonian (1940) compared the action of thiamine and thiamine pyrophosphate on several thiamine-deficient fungi. No significant differences were found in the maximum weights of mycelium formed in the presence of equivalent quantities of these two growth factors. The rate of early growth was greater with thiamine pyrophosphate than with thiamine for Phyco- myces hlakesleeanus and less for Mucor ramannianus and Phytophthora erythroseptica. Pyruvic acid, one of the key intermediate products of carbohydrate metabolism, is transformed into carbon dioxide and acetaldehyde by the action of the enzyme carboxylase. Pyruvic acid accumulates in the culture media of many thiamine-deficient fungi when insufficient thiamine is present. Haag and Dalphin (1940) found that the maximum accumu- lation in Phycomyces hlakesleeanus cultures occurred when about one- twentieth of the optimum amount of thiamine was added. Wirth and Nord (1942) studied the effect of added thiamine upon the accumulation of pyruvic acid in cultures of Fusarium lini, a self-sufficient fungus with respect to thiamine. Some of the data are presented in Table 34. The accumulation of pyruvic acid in the culture medium is common, especially during the early period of growth. Pyruvic acid may be detected qualitatively by adding of iodine solution (KI3) to the culture filtrate and making the solution strongly alkaline with sodium hydroxide. Iodoform is produced instantly without heating. Acetaldehyde, which is very volatile, also reacts with iodine and alkali in the cold to produce iodoform. Sordaria fimicola, Lenzites trahea, or other fungi which produce acid during the early stages of growth may be used to demonstrate the production of pyruvic acid. Specificity. So far as is known, thiamine which occurs in nature has the structure given in the formula. This vitamin has been isolated from only a few substances such as wheat germ and rice polish. The ethyl homologue (ethyl in place of methyl in position 2) of thiamine is slightly more active for certain fungi than ordinary thiamine. Higher homologues 192 PHYSIOLOGY OF THE FUNGI have been reported to be less active or inhibitory. Whether ethyl thia- mine occurs in nature is not known. The student is referred to Schopfer (1943) for further information on thiamine specificity. Table 34. The Effect of Added Thiamine upon the Accumulation of Pyruvic Acid in the Culture Filtrate of Fusarium lint Grown on Glucose- Nitrate Medium (Wirth and Nord, Arch. Biochem. 1, 1942. Published by permission of Academic Press, Inc.) Days of Glucose fermented, g. per liter Pyruvic acid accumulated, mg. per liter Mycelium produced, mg. per 50 ml. incuba- tion Thiamine added, ^g per liter 0 500 0 500 0 500 2 4 6 8 1.5 17.3 34.7 40.9 2.5 13.5 33.9 40.7 50 1,590 1,710 1,550 Trace 80 260 Trace 132 259 91 135 BIOTIN Biotin (vitamin H) was originally isolated as a grow^th factor for yeast. It is known to be the factor which prevents raw-egg-white injury to animals and is the respiratory coenzyme (coenzyme R) for species of Rhizohium. Biotin is active at greater dilutions than are the other vitamins. Pure biotin methyl ester was first isolated by Kogl and Tonnis (1936) w^ho obtained 1.1 mg. of this substance from 250 kg. of dried duck eggs. The structure of biotin was determined by Du Vigneaud et al. (1942a) and confirmed by the synthesis of this compound (Harris et al., 1943). The structure of the biotin molecule is as follows: CO NH NH I I CH CH CH2 CH— (CH2)4— COOH \/ Biotin Some fungi deficient for biotin. Biotin deficiency appears to be characteristic of most yeasts (Burkholder, 1943 ; Burkholder and Moyer, 1943; Leonian and Lilly, 1942). Numerous filamentous fungi have been reported to be deficient for biotin, but this number is not so great as that for thiamine. Frequently biotin deficiency accompanies thiamine deficiency. VITAMINS 193 Among the first investigators to test the action of biotin on filamentous fungi were Kogl and Fries (1937), who showed that Nematospora gossypii did not grow in the absence of biotin. As httle as 0.4 ^g of biotin per liter permitted almost as much growth as did ten times that amount. For most filamentous fungi 5 /xg of biotin per liter is adequate. The effects of biotin deficiency on the development of the ascospores of Sordaria fimicola are shown in Fig. G8. Some filamentous fungi reported as being partially or totally deficient for biotin, with other deficiencies (if any) given in parentheses, are as follows: Chaetomium convolutum (thiamine), Coemansia interrupta (thia- mine), Diplodia macrospora, Endothia parasitica (thiamine), Hypoxylon pruinatum (thiamine), Lamhertella pruni (thiamine), Melanospora destru- ens (thiamine), Memnoniella echinata, Neurospora spp., Ophioholus graminis (thiamine), 0. oryzinus, Ophiostoma catonianum, Penicillium digitatum (thiamine, pyridoxine, pantothenate), Piricularia oryzae (thia- mine), Pleurage curvicolla (thiamine), Podospora curvula (thiamine), Pseudopeziza ribis, Rosellinia arcuata, Sclerotinia camelliae (thiamine, inositol), Sordaria fimicola, Sporormia intermedia, Stachybotrys atra, Thraustotheca clavata. Specificity. The biotin molecule is not separable into moieties as is thiamine. One of the first related compounds to be studied was desthio- biotin. As the name indicates, the molecule no longer contains sulfur. The structure of the desthiobiotin molecule is as follows: CO / \ NH NH I I CH3— CH CH— (CH2)6— COOH Desthiobiotin The removal of sulfur from the biotin molecule destroyed the tetrahydro- thiophene ring and introduced a methyl group. In addition, the acidic chain of desthiobiotin contains one more methylene group than does that of biotin. Stokes and Gunness (1945) tested the growth of some biotin- deficient microorganisms on desthiobiotin and found that this compound was utilized by Neurospora sitophila and three strains of Saccharomyces cerevisiae, but Rhizobium trifolii 209, Lactobacillus casei, and L. arabinosus 17-5 were unable to utilize desthiobiotin. From further experiments it was concluded that the yeast synthesized biotin, or some other compound which replaced it, from desthiobiotin, rather than utilizing desthiobiotin directly. The source of sulfur in the medium was found to influence the amount of desthiobiotin converted into biotin, with methionine and sodium sulfate being better sources than cystine, sulfanilamide, or thiamine thiazole. Lilly and Leonian (1944) studied the effect of desthiobiotin on 45 194 PHYSIOLOGY OF THE FUNGI biotin-deficient microorganisms and found that it replaced biotin for some fungi, while it acted as an antibiotin for some few others. Desthio- biotin replaced biotin quantitatively for Ceratostomella ips. Goldberg et al. (1947) found some homologues of biotin to inhibit growth of yeast 139 and Lactobacillus casei. Whether any of these biotin homologues will replace biotin for other microorganisms must await further testing. These preliminary results indicate that the length of the acidic side chain of the biotin molecule is of great importance in biological activity. Oxybiotin is also known as 0-heterobiotin and has the same structure as biotin except that the sulfur in the tetrahydrothiophene ring has been replaced by oxygen. Pilgrim et al. (1945) found oxybiotin to be active for Lactobacillus casei, L. arabinosus, and a strain of Saccharomyces cere- visiae. Oxybiotin is apparently used as such and is not converted into biotin by the organism (Axelrod et al., 1947). This is the only instance that has come to our attention where a vitamer is used directly instead of being converted into the vitamin. Rubin et al. (1945) had previously reported that oxybiotin was converted into biotin. The cause of this disagreement is unknown. Pimelic acid is a growth factor for certain strains of the diphtheria bacterium (Mueller, 1937). It is reported (Du Vigneaud et al., 1942) that pimelic acid replaced biotin and was probably the precursor in the synthesis of biotin by a strain of the diphtheria organism. The higher and lower homologs of pimelic acid were ineffective. The formula for pimelic acid is HOOC— CH2— CH2— CHa— CH2— CH2— COOH. At present there is no evidence that pimelic acid replaces biotin for the fungi. This observation is supported by the findings of Robbins and Ma (1942), who studied 13 biotin-deficient fungi. A favorable effect of the presence of pimelic acid was reported by Eakin and Eakin (1942), who found that Aspergillus niger synthesizes much more biotin in the presence of pimelic acid than in its absence. Cysteine and also cystine increase the synthesis of biotin. The lower homologues of pimelic acid (adipic, glutaric, and succinic) were without effect, while the higher homologs (suberic and azelaic) were as effective as pimelic acid. This is interesting, inasmuch as homobiotin and bishomobiotin are reported inactive for yeast growth (Goldberg et al., 1947). Mode of action. It has been assumed that biotin acts as a coenzyme for various enzyme systems, but definite proof seems to be lacking, Winzler et al. (1944) found that, when biotin was added to a biotin- starved yeast, some time elapsed before any effect was noted. The order of response was fermentation, respiration, and growth. The assimilation of ammonia did not take place unless biotin was added. The presence of aspartic acid in the culture medium has been shown to reduce the amount of biotin required by Torula cremoris (Koser et al., VITAMINS 195 1942) and by Memnoniella echinata and Stachyhotrys atra (Perlman, 1948). There is also evidence (Stokes et al., 1947) that biotin plays a role in the synthesis of aspartic acid by certain bacteria. Thus, it appears probable that one of the functions of biotin is connected with the synthesis of aspartic acid. When aspartic acid is added to the medium, it is unnecessary for the organism to perform this synthesis and the need for biotin is greatly reduced. However, it should be noted that, although the absolute amount of biotin needed is reduced, exogenous biotin is still required by these biotin-deficient organisms. From this it may be deduced that biotin has a multiple role in the cell. INOSITOL meso-Inositol (also known as inactive inositol, isoinositol, inosite, or dambose) is widely distributed in both plants and animals. It was first isolated in 1850. It was not until 1928 that Eastcott (1928) showed that it was a growth factor for a strain of yeast. Later, Woolley (1940) recognized it as a vitamin for animals. meso-Inositol is a hexahydroxy- cyclohexane. It has the following configuration: H H Q Q OH/i i\ H 1/ OH 0H\| c c :\ H OH/1 H \| 1/ OH C C OH li meso-Inositol There are seven different cis-trans isomers, which are optically inactive, and a pair of optically active d and I forms. The available evidence indicates that the stereochemical configuration of weso-inositol is specific for vitamin activity. Some of the isomers have only slight activity. Inositol is active only in high concentrations as compared to the other vitamins. The usual amount added is around 5 mg. per hter of medium. Fungi deficient for inositol. Many strains of yeast are deficient for this vitamin, while others are not. In most cases the deficiency appar- ently is partial rather than total. Partial deficiencies for various yeasts are reported by Leonian and Lilly (1942), Burkholder (1943), and Burk- holder and Moyer (1943). In the last two references total deficiencies for inositol are reported for Saccharomyces uvarum Y 969 and Schizosac- charomyces pomhe. Kogl and Fries (1937) were apparently the first to investigate the action of inositol on various filamentous fungi. They found that Nemato- spora gossypii was totally deficient and that Lophodermium pinastri was 196 » PHYSIOLOGY OF THE FUNGI partially deficient for this vitamin. The partial deficiency of Sclerotinia camelliae is shown in Fig. 35. Deficiencies for inositol are commonly accompanied by deficiencies for thiamine and biotin. Trichophyton discoides is reported as being totally deficient for inositol, pyridoxine, and thiamine (Robbins et at., 1942). Totally deficient mutants of Neurospora crassa have been developed. Their use in bioassays for inositol was described by Beadle (1944) and by Leonian and Lilly (1945). Some filamentous fungi reported to be partially or totally deficient for inositol, with other deficiencies given in parentheses, are as follows: Colletotrichum lindemuthianum (certain strains only), Epichloe typhina (thiamine), Lophodermium pinastri (thiamine, biotin), Melanconium hetulinum (thiamine, biotin), Nematospora gossypii (thiamine, biotin), Sclerotinia camelliae (thiamine, biotin), Trichophyton discoides (thiamine, pyridoxine), Valsa pini (thiamine, biotin). The effects of temperature upon the synthesis of inositol by Sclerotinia camelliae and upon the toxicity of high concentrations of inositol at high temperatures were described by Barnett and Lilly (1948) and are illus- trated in Fig. 42. Mode of action. The addition of inositol overcame the inhibition of growth of Rhizopus suinus due to excess thiamine (Schopfer and Guilloud, 1945). In part, the inhibition was due to an increased production of alcohol (pyruvate ^ acetaldehyde—^ alcohol). Similarly, we have observed in our laboratory the same favorable effect of inositol on growth of certain fungi which are inhibited by the presence of excess thiamine. NICOTINIC ACID A deficiency for nicotinic acid, or nicotinic acid amide, leads to pellagra in man and blacktongue in dogs. The structural formulas of these com- pounds follow: /\^r.f^c^vi /\ -CONH2 Nicotinic acid Nicotinic acid amide Nicotinic acid was obtained by the oxidation of nicotine in 1867. Knight (1937) and Mueller (1937o) recognized that nicotinic acid amide was a growth factor for certain bacteria. So far as is known, the amide is the form utilized by organisms. Some microorganisms can convert nicotinic acid into its amide with ease, others with difficulty; still others are unable to use nicotinic acid but require either nicotinic acid amide or a coenzyme containing the amide. Fungi deficient for nicotinic acid. Rogosa (1943) tested 114 strains of yeast that ferment lactose and found that all of them required an exoge- VITAMINS 197 nous supply of nicotinic acid for growth. Rogosa used the technique of serial passage in a medium devoid of nicotinic acid. It is possible to overlook a vitamin deficiency by failure to observe this precaution. Yeasts found to be deficient for this vitamin include Saccharomyces anamensis 154, S. lactis 131, S. fragilis 15, Zy go saccharomyces lactis (two strains), Torida lactosa 168, T. sphaerica 13, T. cremoris 2, Torulopsis kefyr 149, Mycotorula lactis 130. Strains of Saccharomyces cerevisiae failed to show deficiency for nicotinic acid (Rogosa, 1943; Leonian and Lilly, 1942; Burkholder, 1943). Until recently, nicotinic acid deficiency among filamentous fungi iso- lated from nature was unknown. Cantino (1948) has shown that Blasto- cladia pringsheimii is completely deficient for nicotinamide and partially deficient for thiamine and biotin. Some of Cantino's results are pre- sented in Fig. 37. A second filamentous fungus, a strain of Microsporum, audouini, is reported as deficient for nicotinic acid (Area Leao and Cury, 1949). Mutants deficient for this vitamin have been developed in Neurospora by Bonner and Beadle (1946) and in Penicillium by Bonner (1946). Specificity. In so far as the fungi are concerned, nicotinic acid replaces nicotinic acid amide, but few critical studies in this connection have been made. Various studies have been made of the specificity for bacteria of the compounds related to nicotinic acid. Bovarnick (1943) reported that heating asparagine and glutamic acid together produced a compound which replaced nicotinic acid or its amide for various species of bacteria. This author later showed that this substance was nicotinic acid amide. This is an unsuspected way of adding a vitamin to a basal medium. Mode of action. Nicotinic acid amide is a constituent of two or more coenzymes. Codehydrogenase I on hydrolysis yields adenine, nicotinic acid amide, and two molecules of D-ribosephosphoric acid. Codehydro- genase II yields the same products as codehydrogenase I except that three molecules of phosphoric acid, instead of two, are produced. In the literature codehydrogenase I is often referred to as DPN (diphospho- pyridine nucleotide) and codehydrogenase II as TPN (triphosphopyridine nucleotide). These coenzymes in combination with specific proteins form enzyme systems which transfer hydrogen (oxidation-reduction). Apparently the amide of nicotinic acid is reversibly oxidized and reduced in the process. One organism. Hemophilus parainfluenzae, requires codehydrogenase I as a growth factor. This organism is unable to form the coenzyme u'hen furnished with the moieties, nicotinic acid amide, adenine, D-ribose, and phosphate. DPN is also known as factor V (Gingrich and Schlenk, 1944). Other bacteria are known which require preformed coenzymes as growth factors. While no fungus isolated from nature has yet been 198 PHYSIOLOGY OF THE FUNGI shown to require such growth factors, it is possible that some do exist. Such requirements may be found among the artificially induced mutants. PANTOTHENIC ACID Pantothenic acid was first discovered (Williams et al., 1932) as a growth factor for the Gebriide Mayer strain of Saccharomyces cerevisiae. The isolation, identification, and synthesis of this compound was complete by 1940. It was later shown to be a vitamin for animals. Pantothenic acid consists of two moieties joined together by means of an amide link- age. The chemical formula for this vitamin is given below: CH3 I HO— CH2— C— CHOH— CO— NH— CH2— CH2— COOH I CH3 Pantothenic acid Pantothenic acid may be hydrolyzed to form /3-alanine (jS-amino- propionic acid), the formula of which is H2N — CH2 — CH2 — COOH, and a,7-dihydroxy-/3,;5-dimethylbutyric acid, a substituted butyric acid that forms a lactone by elimination of one molecule of w^ater between the carboxyl and the gamma hydroxyl (pantoyl lactone). Pantothenic acid is thus analogous to thiamine, in that the molecule may be split into two moieties. We might expect to find different pantothenic acid-deficient organisms which require the intact molecule or one or both moieties. It was found that the Gebriide Mayer strain of Saccharomyces cerevisae was stimulated by /3-alanine and that this yeast completed the synthesis of pantothenic acid when furnished with )8-alanine in the medium (Wein- stock et al., 1939). Most yeasts deficient for pantothenic acid are unable to synthesize the j8-alanine moiety of this vitamin. In general, this moiety is not used so efficiently as pantothenic acid, and more than an equivalent amount is required to support the same amount of growth. The composition of the medium affects utilization, since, in the presence of sufficient asparagine, /3-alanine is not utilized (Atkin et al., 1944). So far as is known, none of the fungi require pantoyl lactone as a growth factor, but this compound was found (Ryan et al., 1945) to replace panto- thenic acid for Clostridium septicum. It was shown by microbiological tests that this bacterium completed the synthesis of pantothenic acid. Fungi deficient for pantothenic acid. Of the 10 strains of Saccharo- myces cerevisiae tested for vitamin deficiencies by Leonian and Lilly (1942), 9 w^ere highly deficient for this vitamin. Burkholder (1943) found 14 of the 38 species and strains tested to be deficient for pantothenic acid, 9 of these being species of Saccharomyces. It appears that deficiency for this vitamin is more common in Saccharomyces than in other genera of yeasts. Varying degrees of pantothenic acid deficiency were found in VITAMINS 199 species of Zygosaccharomyces (Lockhead andLanderkin, 1942). /S-Alanine could be used in place of pantothenic acid for the deficient species of Zygosaccharomyces. Growth of Penicillium digitatum is reported (Wooster and Cheldehn, 1945) to be stimulated by pantothenate, as well as by pyridoxine, biotin, and thiamine. To our knowledge this is the only report of a filamentous fungus isolated from nature being stimulated by the presence of this vitamin. Tatum and Beadle (1945) reported a mutant of Neurospora which was deficient for pantothenic acid. Specificity. As in the case of thiamine and inositol, the structure of pantothenic acid is almost specific for activity. A hydroxypantothenic acid synthesized by Mitchell et al. (1940) has a varying ability to replace pantothenic acid for some organisms. The activity of this compound for the Gebriide Mayer yeast was low as compared with pantothenic acid. Mode of action. Pantothenic acid was found to favor the accumula- tion of glycogen by yeasts (Williams et al, 1936), and to increase markedly the rate of carbon dioxide evolution by the pantothenic acid-deficient Gebriide Mayer yeast (Pratt and Williams, 1939). More recent work (Novelli and Lipmann, 1947) has show^n that pantothenic acid is phos- phorylated and acts as a coenzyme. This enzyme system performs vari- ous oxidations and acetylations in the cell. PYRIDOXINE Pyridoxine is also known as adermin or as vitamin Be. While inositol and pantothenic acid were first investigated as growth factors for micro- organisms, pyridoxine was discovered as a result of animal research. This vitamin was isolated independently by five groups of workers in 1938 and was synthesized the next year. The structural formula is given below : HO- CHs- ■^ n— CH2OH Pyridoxine Pyridoxine is quite soluble in water and is stable to acid and alkah but is destroyed by light. Fungi deficient for pyridoxine. Partial or total deficiencies for this vitamin have been reported for various species and strains of yeasts (Eakin and Williams, 1939; Burkholder, 1943). Among these are Sac- charomyces carlsbergensis var. mandshuricus Y-379, S. chodati Y-140, S. oviformis, S. ludwigii, and Mycoderma valida Y-7. Leonian and Lilly (1942) found that the omission of either thiamine or pyridoxine alone from the medium was without effect on 9 of the 10 strains 200 PHYSIOLOGY OF THE FUNGI of yeast tested. However, the omission of both pyridoxine and thiamine caused a decrease in the growth of two of these strains. Apparently these two yeasts were capable of synthesizing either thiamine or pyri- doxine, provided that the other vitamin w^as present. This is a common effect among fungi partially deficient for two or more vitamins. The presence of one vitamin for which a fungus is partially deficient may enable the fungus to synthesize other vitamins with greater ease. Among the filamentous fungi, deficiency for pyridoxine seems to be characteristic of certain species of Ceratostomella and a few other fungi (Robbins and Ma, 1942o, 19426). Some species reported to be deficient for pyridoxine, with other deficiencies given in parentheses, are Cerato- stomella ulmi, C. ips (thiamine, biotin), C. pseudotsugae (thiamine), C. piceaperda 240 (biotin), C. pini (thiamine, biotin), C montium (thiamine, biotin), C. pilifera, C. multiannulata (thiamine), C. pluriannulata (thia- mine), C. microspora (thiamine, biotin), Ophiostoma catonianum (thia- mine), Trichophyton discoides (thiamine, inositol). Specificity. One of the important uses of vitamin-deficient organisms is for the purpose of vitamin assay. Certain vitamin-deficient fungi and bacteria are used to determine the vitamin content of foodstuffs and other natural products. For such tests to be of any value, it is necessary to know if the organism used responds to substances other than the vitamin itself. Snell et at. (1942) found that Streptococcus faecalis gave much greater apparent yields of pyridoxine when used for assay than did yeast. It was then discovered (Snell, 1942) that autoclaving pyridoxine with the basal medium for 20 min. increased the activity of pyridoxine forty-one times, and that this change in activity for certain organisms was correlated with oxidation and heating with certain amino acids. Snell (1944) then postulated that vitamers of pyridoxine were formed by these treatments. When this problem was under investigation, these vitamers of unknown structure were called "pseudopyridoxine," which was later found to consist of either one or both of the following compounds : HO- CH3 ^^— CHoOH HO— r^^— CH2OH Pyridoxal Pyridoxamine These two compounds were synthesized by Harris et al. (1944) and tested by Snell. It was concluded that this vitamin consists of three or more closely related compounds. Saccharomyces carlsbergensis responds about equally to the three compounds, while the reaction of certain bacteria is much greater to pyridoxal and pyridoxamine than to pyridoxine, Ceratosto- VITAMINS 201 mella ulmi grew at a more rapid rate with pyridoxamine than vvith pvri- doxal or pyridoxine (Snell and Rannefelt, 1945). All three forms of this vitamin occur in natural products. Assays for this vitamin are discussed in Chap. 10. Mode of action. One of the earliest clues to the action of pyridoxine was discovered by Snell and Guirard (1943) in the interrelationship among glycine, alanine, and pyridoxine and growth of Streptococcus faecalis R. They found that alanine could replace pyridoxine for this organism and that glycine caused inhibition which was overcome by the addition of either alanine or pyridoxine. It was also found that /3-alanine, serine, and threonine inhibited growth. It is possible that alanine serves as a precursor for pyridoxine in this organism, or that one function of pyridoxine is the synthesis of alanine. At any rate the action of pyridoxine appears to be connected with either amino-acid synthesis or amino-acid utilization, or both. Like other vitamins, pyridoxine (or its conversion products) has been assumed to function in the cell as a part of a coenzyme. Pyridoxal is phosphorylated before it functions in enzyme systems. In this it is like thiamine and pantothenic acid. Pyridoxal phosphate is said to function as a coenzyme in the transformation of tryptophane into indole by Escherichia coli (Wood et at., 1947). We may assume that the function of this vitamin is the same in the fungi as in the bacteria. p-AMINOBENZOIC ACID p-Aminobenzoic acid has the following structure: COOH NH2 p-Aminobenzoic acid Rubo and Gillespie (1940) found p-aminobenzoic acid to be a growth factor for nine strains of Clostridium acetohutylicum. Most of the interest in this compound centers in its antagonistic action to sulfonamides. A discussion of this subject is presented in Chapter 11. Fungi deficient for p-aminobenzoic acid. Robbins and Ma (1944) reported Rhodotorula aurantica to be deficient for p-aminobenzoic acid and thiamine. Concentrations of as low as 0.03 /xg per liter had a positive effect on the growth of this yeast, while maximum growth was attained in the presence of 3 /zg per liter. The intensity of the pink color developed by this yeast was a function of the p-aminobenzoic acid content of the medium. 202 PHYSIOLOGY OF THE FUNGI In so far as we are aware, no filamentous fungus isolated from nature has been shown to be deficient for p-aminobenzoic acid. Tatum and Beadle (1942) described a mutant of Neurospora which was unable to synthesize this vitamin. Wyss et at. (1944) found that the availability of p-aminobenzoic acid to the deficient mutant of Neurospora crassa was a function of the pH of the medium (see Fig. 41). Mode of action. The functions of p-aminobenzoic acid are unknown. We may assume, on the basis of the behavior of other vitamins, that it functions as a coenzyme, or as a part of a coenzyme. Recent work indicates that p-aminobenzoic acid is a constituent part of folic acid. RIBOFLAVIN The structure of riboflavin is given below: CH2OH I HC— OH I HC— OH I HC— OH CH2 I N N \c^ \c=o N C s Riboflavin Many bacteria, especially species of Lactobacillus are unable to synthe- size riboflavin (Peterson and Peterson, 1945). So far as we are able to determine, none of the fungi isolated from nature have been found to be deficient for riboflavin. This vitamin is synthesized by the fungi. Mitchell and Houlahan (1946) described a mutant of Neurospora which required the addition of riboflavin to the medium for growth at tempera- tures above 28°C. Between 15 and 25°C. the growth rate of the mutant without added riboflavin was equal to that of the wild type. The rate of growth decreased rapidly as the temperature increased from 25 to 28°C. SUMMARY It is assumed that all living organisms require a number of vitamins, or growth factors, for normal growth, reproduction, and other vital proc- esses. However, organisms differ widely in their synthetic capacities for the various vitamins. Some fungi are self-sufficient with respect to vitamins, being able to synthesize their vitamins from pure chemicals of a synthetic medium. Others lack the ability to synthesize sufficient VITAMINS 203 quantities of one or more vitamins and are called vitamin-deficient fungi. The deficiency may be single or multiple, complete or partial. Partial deficiency may vary from nearly complete to nearly self-suflficient and is more pronounced during the early stages of growth. A single deficiency for thiamine has been more commonly reported among filamentous fungi than any other type. Biotin deficiency is like- wise commonly found, often in combination with thiamine deficiency. Deficiencies for inositol and pyridoxine are less common. Two filamen- tous fungi isolated from nature are reported to be deficient for nicotinic acid. Numerous other deficiencies have been induced in mutants by ir- radiation. Some yeasts show complete or partial multiple deficiencies for three to six vitamins, while relatively few filamentous fungi are deficient for as many as three vitamins. Absolute deficiencies are not known to be influenced by the environ- ment, while conditioned deficiencies may be affected either by nutritional factors or by factors of the physical environment. Among these, tem- perature and the composition and pH of the medium seem to be the most important. Methods of detecting vitamin deficiencies are exact, and accurate determination depends on the ability or inability of a fungus to grow on a synthetic medium composed of pure chemicals, to which known amounts of the various vitamins to be tested are added. Vegetative growth, measured by dry weight, is apparently the most useful criterion of the utilization of vitamins, although reproduction and other processes are likewise affected. Compounds having vitamin activity but differing in molecular struc- ture are called vitamers. In general, only compounds of closely related structure have vitamin activity. The inhibitory effects of vitamins in excess quantities are apparently common. They are usually evident by slight reduction in rate or maxi- mum amount of growth and are more common with self-sufficient fungi than with those deficient for the vitamin in question. Thiamine is more commonly reported as a growth depressor than other vitamins. One instance of severe inhibition due to excess inositol and temperatures near maximum for growth is discussed. The known effects of vitamins on the growth of fungi emphasize the important fact that growth is a result of a number of interacting factors, among which are the vitamins. A proper balance between the different vitamins and with the other nutritional and environmental factors must exist if maximum rate of growth is to take place. REFERENCES Ar^a Leao, a. E., and A. Cury: Sobre a exigencia de ^cido nicotlnico observada em cogumelo filamentoso; {Microsporum audouini), O Hospital (Rio de Janiero) 35: 347-351, 1949. 204 PHYSIOLOGY OF THE FUNGI Atkin, L., W. L. Williams, A. S. Schultz, and C. N. Frey: Yeast microbiological methods for determination of vitamins. Pantothenic acid, Ind. Eng. Chern., Anal. Ed. 16: 67-71, 1944. AxELROD, A. E., B. C. Flinn, and K. Hofmann: The metabolism of oxybiotin in yeast, Jour. Biol. Chem. 169 : 195-202, 1947. Barnett, H. L., and V. G. Lilly: The interrelated effects of vitamins, temperature and pH upon vegetative growth of Sclerotinia camelliae, Am. Jour. Botany 35: 297-302, 1948. Beadle, G. W. : An inositolless mutant strain of Neurospora and its use in bioassays, Jour. Biol. Chem. 156 : 683-689, 1944. Bonner, D.: Production of biochemical mutations in Penicillium, Ann. Jour. Botany 33: 788-791, 1946. Bonner, D., and G. W. Beadle: Mutant strains of Neurospora requiring nicotin- amide or related compounds for growth, Arch. Biochem. 11 : 319-328, 1946. Bovarnick, M. R. : Substitution of heated asparagine-glutamate mixture for nicotinamide as a growth factor for Bacterium dysenteriae and other micro- organisms. Jour. Biol. Chem. 148: 151-161, 1943. BuBK, D., M. L. Hesselback, D. F. MacNeary, and R. J. Winzler: The vitamer concept. Trans. N.Y. Acad. Sci. 6: 275-283, 1944. *BuRKHOLDER, P. R. : Vitamin deficiencies in yeasts. Am. Jour. Botany 30: 206-211, 1943. BuRKHOLDER, P. R., I. McVeigh, and D. Moyer: Studies on some growth factors of yeasts. Jour. Bad. 48: 385-391, 1944. Burkholder, p. R., and D. Moyer: Vitamin deficiencies of fifty yeasts and molds, Bull. Torrey Botan. Club 70 : 372-377, 1943. Canting, E. C.: The vitamin nutrition of an isolate of Blastocladia pringsheimii, Am. Jour. Botany 35 : 238-242, 1948. DU Vigneaud, v., K. Dittmer, E. Hague, and B. Long: The growth-stimulating effect of biotin for the Diphtheria bacillus in the absence of pimelic acid, Science 96: 186-187, 1942. DU Vigneaud, V., K. Hofmann, and D. B. Melville: On the structure of biotin. Jour. Am. Chem. Soc. 64 : 188-189, 1942a. Eakin, R. E., and E. A. Eakin: A biosynthesis of biotin. Science 96: 187-188, 1942. Eakin, R. E., and R. J. Williams: Vitamin Be as a yeast nutrilite. Jour. Am. Chem. Soc. 61: 1932, 1939. Eastcott, E. v.: Wildiers' Bios. The isolation and identification of "Bios 1," Jour. Phys. Chem. 32: 1094-1111, 1928. Elliott, E. S. : Effect of vitamins on growth of some graminicolous species of Helminthospori um and Fusarium, Proc. West Va. Acad. Sci. 20: 65-68, 1949. *Fries, N.: The nutrition of fungi from the aspect of growth factor requirements, Trans. Brit. Mycol. Soc. 30: 118-134, 1948. Fromageot, C., and J. L. Tschang: Sur la synthese des pigments carotenoides par Rhodotorula sanniei, .Arch. Mikrobiol. 9 : 434-448, 1938. Gingrich, W., and F. Schlenk: Codehydrogenase I and other pyridinium com- pounds as V-factor for Hemophilus influenzae and H. parainfluenzae, Jour. Bad. 47 : 535-550, 1944. Goldberg, M. W., L. H. Sternbach, S. Kaiser, S. D. Heineman, J. Scheiner, and S. H. Rubin: Antibiotin effect of some biotin homologs, Arch. Biochem. 14:480-482, 1947. Haag, E., and C. Dalphin: Une Reaction biochimique de I'aneurine d'une grande sensibility, Compt. rend. soc. phys. hist. nat. Geneve 57 : 76-77, 1940. VITAMINS 205 Harris, S. A., D. Heyi, and K. Folkers: The structure and synthesis of pyridox- amine and pyridoxal, Jour. Biol. Chem. 154: 315-316, 1944. Harris, S. A., D. E. Wolf, R. Mozingo, and K. Folkers: Synthetic biotin, Science 97:447-448, 1943. Knight, B. C. J. G.: The nutrition of Staphylococcus aureus: Nicotinic acid and vitamin Bi, Biochem. Jour. 31: 731-737, 1937. KoGL, F., and N. Fries: Ueber den Einfiuss von Biotin, .Aneurin und Meso-Inosit auf das Wachstum verschiedener Pilzarten, Zeit. physiol. Chem. 249: 93-110, 1937. KoGL, F., and B. Tonnis: Ueber das Bios-Problem. Darstelking von krystal- lisiertem Biotin aus Eigelb, Zeit. physiol. Chem. 242 : 43-73, 1936. KosER, S. A., M. H. Wright, and A. Dorfman: Aspartic acid as a partial substitute for the growth stimulating effect of biotin on Torula cremoris, Proc. Soc. Exptl. Biol. Med. 51 : 204-205, 1942. Leonian, L. H., and V. G. Lilly: Auxithals synthesized by some filamentous fungi, Plant Physiol. 15 : 515-525, 1940. Leonian, L. H., and V. G. Lilly: The effects of vitamins on ten strains of Sac- charomyces cerevisiae, Am. Jour. Botany 29: 459-464, 1942. Leonian, L. H., and V. G. Lilly: The comparative value of different test organisms in the microbiological assaj^ of B vitamins, West Va. Agr. Expt. Sta. Bull. 319, 1945. Lilly, V. G., and H. L. Barnett: The influence of pH and certain growth factors on mycelial growth and perithecial formation by Sordaria fimicola, Amer. Jour. Botany 34: 131-138, 1947. Lilly, V. G., and H. L. Barnett: The inheritance of partial thiamine deficiency in Lenzites trabea, Jour. Agr. Research 77: 287-300, 1948. Lilly, V. G., and L. H. Leonian: The growth rate of some fungi in the presence of co-carboxylase, and the moieties of thiamin, Proc. West Va. Acad. Set. 14 : 44-49, 1940. Lilly, V. G., and L. H. Leonian: The anti-biotin effect of desthiobiotin, Science 99: 205-206, 1944. LocKHEAD, A. G., and G. B. Landerkin: Nutrilite requirements of osmophilic yeasts. Jour. Bad. 44: 343-351, 1942. Mathur, R. S., H. L. Barnett, and V. G. Lilly: Sporulation of Colletotrichum. lindemuthianum in culture. Phytopathology 40: 104-114, 1950. Mitchell, H. K., and M. B. Houlahan: Neurospora. IV. A temperature-sensi- tive riboflavinless mutant, Am. Jour. Botany 33: 31-35, 1946. Mitchell, H. K., E. E. Snell, and R. J. Williams: Pantothenic acid. IX. The biological activity of hydroxy-pantothenic acid, Jour. Am. Chem. Soc. 62 : 1791-1792, 1940. Mueller, J. H.: Studies on cultural requirements of bacteria. X. Pimclic acid as a growth stimulant for C. diphtheriae, Jour. Bad. 34: 163-178, 1937. Mueller, J. H. : Nicotinic acid as a growth accessory substance for the diphtheria bacillus. Jour. Bad. 34: 439-441, 1937a. NovELLi, G. D., and F. Lipmann: Bacterial conversion of pantothenic acid into co-enzyme A (acetylation) and its relation to pyruvic oxidation. Arch. Biochem. 14:23-27, 1947. Perlman, D.: On the nutrition of Memnoniella echinata and Stachybotrys atra, Am. Jour. Botany 35: 36-41, 1948. Peterson, W. IL, and M. S. Peterson: Relation of bacteria to \dtamins and other growth factors, Bact. Revs. 9 : 49-109, 1945. 206 PHYSIOLOGY OF THE FUNGI Pii.GRiM, F. J., A. E. AxELROD, T. WiNNicK, and K. Hofmann: The microbiological activity of an oxygen analog of biotin, Science 102 : 35-36, 1945. Pratt, E. F., and R. J. Williams: The effects of pantothenic acid on respiratory activity. Jour. Gen, Physiol. 22 : 637-647, 1939. RoBBiNS, W. J., and F. Kavanagh: Thiamin and growth of Pythium butleri, Bull. Torrey Botan. Club 65: 453-461, 1938. RoBBiNS, W. J., and F. Kavanagh: Thiazole effect on Phycomyces, Proc. Natl. Acad. Sci. U.S. 27: 423-427, 1941. ♦RoBBiNS, W. J., and V. Kavanagh: Vitamin deficiencies of the filamentous fungi, Botan Rev. 8: 411-471, 1942. RoBBiNS, W. J., and R. Ma: Pimelic acid, biotin and certain fungi, Science 96: 406-407, 1942. *RoBBiNS, W. J., and R. Ma: Vitamin deficiencies of Ceratostomella and related fungi, Am. Jour. Botany 29: 835-843, 1942a. RoBBiNS, W. J., and R. Ma: Vitamin deficiencies of Ceratostomella, Bull. Torrey Botan. Club 69: 184-203, 19426. RoBBiNS, W. J., and R. Ma: A Rhodotorula deficient for para-aminobenzoic acid, Science 100 : 85-86, 1944. RoBBiNS, W. J., J. E. MacKinnon, and R. Ma: Vitamin deficiencies of Trichophyton discoides, Bull. Torrey Botan. Club 69: 509-521, 1942. RoGOSA, M.: Nicotinic acid requirements of certain yeasts, Jour. Bact. 46: 435-440, 1943. Rosenberg, H. R. : Chemistry and Physiology of the Vitamins, Interscience Pub- lishers, Inc., New York, 1942. Rubin, S. H., D. Flowers, F. Rosen, and L. Drekter: The biological activity of 0-heterobiotin, Arch. Biochem. 8: 79-90, 1945. RuBBO, S. D., and J. M. Gillespie: Para-aminobenzoic acid as a bacterial growth factor, Nature 146 : 838-839, 1940. Ryan, F. J., R. Ballentine, E. Stolong, M. E. Corson, and L. K. Schneider: The biosynthesis of pantothenic acid, Jour. Am. Chein. Soc. 67 : 1857-1858, 1945. ScHOPFER, W. H.: Ueber die Wirkung von reinen kristallisierten Vitaminen auf Phycomyces, Ber. d. deut. botan. Ges. 52: 308-311, 1934. ScHOPFER, W. H.: Recherches sur le metabolisme de I'azote d"un microorganisme acellulaire (Phycomyces blakesleeanus Bgf.). Le role des facteurs de croissance, Protoplasma 28: 381-434, 1937. *ScHOPFER, W. H.: Plants and Vitamins, Chronica Botanica Co., Waltham, 1943. ScHOPFER, W. H., and M. Guilloud: Recherches experimentales sur les facteurs de croissance et le pourvoir de synthese de Rhizopus cohnii Berl. et de Toni (Rhizo- pus suinus Neilson), Zeit. fur Vitaminforsch. 16: 181-296, 1945. Snell, E. E. : Effect of heat sterilization on growth-promoting activity of pyridoxine for Streptococcus lactis R., Proc. Soc. Exptl. Biol. Med. 51: 356-358, 1942. Snell, E. E.: The vitamin activities of "pyridoxal" and "pyridoxamine," Jour. Biol. Chem. 154: 313-314, 1944. Snell, E. E., and B. M. Guirard: Some interrelationships of pyridoxine, alanine and glycine in their effect on certain lactic acid bacteria, Proc. Natl. Acad. Sci. U.S. 29: 66-73, 1943. Snell, E. E., B. M. Guirard, and R. J. Williams: Occurrence in natural products of a physiologically active metabolite of pyridoxine, Jour. Biol. Chem. 143 : 519-530, 1942. Snell, E. E., and A. N. Rannefelt: The vitamin Be group. III. The vitamin activity of pyridoxal and pyridoxamine for various organisms, Jour. Biol. Chem. 157 : 475-489, 1945. VITAMINS 207 Stokes, J. L., J. W. Foster, and H. B. Woodward: Synthesis of pyridoxin by a "pyridoxin-less" x-ray mutant of Neurospora sitophila, Arch. Biochem. 2: 235-245, 1943. Stokes, J. L., and M. Gunness: Microbiological activity of synthetic biotin, its optical isomers and related compounds, Jour. Biol. Chem. 157: 121-126, 1945. Stokes, J. L., A. Larsen, and M. Gunness: Biotin and the synthesis of aspartic acid by microorganisms. Jour. Biol. Chem. 167: 613-614, 1947. Strauss, B. S.: Studies on the Be-requiring, pH-sensitive mutants of Neurospora crassa, Arch. Biochem. 30: 292-305, 1951. Tatum, E. L., and G. W. Beadle: Genetic control of biochemical reactions in Neurospora: an "aminobenzoic-less" mutant, Proc. Natl. Acad. Sci. U.S. 28: 234-243, 1942. Tatum, E. L., and G. W. Beadle: Biochemical genetics of Neurospora, Ann. Mis- souri Botan. Garden 32 : 125-129, 1945. Weinstock, H. H., H. K. Mitchell, E. F. Pratt, and R. J. Williams: Pantothenic acid. IV. Formation of beta-sAanme by cleavage. Jour. Am. Chem. Soc. 61: 1421-1425, 1939. Williams, R. J., C. M. Lyman, G. H. Goodyear, and J. H. Truesdail: Is the nutrilite for Gebriide Mayer yeast of universal biological importance?, Jour. Am. Chem. Soc. 54: 3462-3463, 1932. Williams, R. J., W. A. Mosher, and E. Rohrman: The importance of "panto- thenic acid" in fermentation, respiration and glycogen storage, Biochem. Jour. 30: 2036-2039, 1936. Williams, R. R., and T. D. Spies: Vitamin Bi (Thiamin) and Its Use in Medicine, The Macmillan Company, New York, 1938. WiNZLER, R. J., D. Burk, and V. du Vigneaud: Biotin in fermentation, respiration and nitrogen assimilation by yeast. Arch. Biochem. 6 : 25-47, 1944. WiRTH, J. C, and F. F. Nord: Essential steps in the enzymatic breakdown of hexoses and pentoses. Interaction between dehydrogenation and fermentation, Arch. Biochem. 1: 143-163, 1942. Wood, W. A., I. C. Gunsalus, and W. W. Umbreit: Function of pyridoxal phos- phate : Resolution and purification of the tryptophanase enzyme of Escherichia coli, Jour. Biol. Chem. 170: 313-321, 1947. Woolley, D. W.: The nature of the antialopecia factor, Science 92: 384-385, 1940. Wooster, R. C., and V. H. Cheldelin: Growth requirements of Penicillium digita- tum, Arch. Biochem. 8: 311-320, 1945. Wyss, O., V. G. Lilly, and L. H. Leonian: The effect of pH on the availability of p-aminobenzoic acid to Neurospora crassa, Science 99 : 18-19, 1944. CHAPTER 10 FUNGI AS TEST ORGANISMS Numerous physiological problems are accessible to investigation through the use of microorganisms. By the proper choice of deficient organisms, it is feasible to detect minute amounts of physiologically active compounds such as the vitamins and amino acids. Knowledge has been gained of the way vitamins and amino acids are synthesized and destroyed by various organisms. The amino-acid composition of proteins and the availability of certain essential elements in soil may be determined by the use of fungi and bacteria. These highly practical studies are based upon a knowledge of the compounds and elements essential for the nutrition of microorganisms. Since these are, in general, the same elements and compounds needed by animals, there is a very close relation between fungus and animal physiology in nutritional problems. Foodstuffs for man and animals are the most common materials analyzed in routine assays. Some of the advantages which have contributed to the widespread use of microorganisms for assay purposes are simple technique and apparatus, sensitivity, specificity, and the short time required. Perhaps the most important single factor is the small sample needed and the fact that little or no purification or concentration of the active material is required. These advantages are to be compared with chemical methods or the use of animals for obtaining the same information. All analytical methods have advantages and disadvantages. A knowledge of the limitations of any method is essential for valid results. Most microbiological assays depend upon the proportional response of deficient test organisms to the substances for which they are deficient. This proportional response occurs only for a limited range of concentra- tions. The usable range of concentration depends upon the substance being assayed, the test organism, and the basal medium. In theoiy, any organism may be used to assay any substance for which it is deficient, but in practice not all organisms having the same deficiency are equally suitable. For example, Rhizohium trijolii 205 is about 100 times as sen- sitive to biotin as Sordaria fimicola. The following are essential to any quantitative microbiological assay: (1) a suitable test organism; (2) the preparation of a basal medium ade- quate in all respects, but essentially free from the substance to be assayed ; (3) liberation, from the material to be analyzed, of the substance to be 208 FUNGI AS TEST ORGANISMS 209 assayed, in a water-soluble condition; (4) a standard sample of the sub- stance to be analyzed ; (5) preparation of a range of concentrations of the known and unknown substances in the basal medium ; (6) uniform inocula- tion; (7) incubation under uniform conditions; (8) measuring the response of the test organism; (9) construction of the standard curve from the response of the test organism to kno\\Ti amounts of the substance under test; (10) calculating the content of the substance contained in the sample. The above discussion assumes the use of pure compounds in obtaining standard curves. The utility of microbiological assay methods is not confined to the assay of known compounds. They are of great utihty in studies of methods of isolation of new growth factors and other active compounds. These occur in complex natural products and, before they are isolated, are known only by the physiological effects they produce in living organisms. Given a deficient fungus, or other organism, it is pos- sible to follow the efficiency of the various steps in an isolation procedure. The isolation of many of the water-soluble vitamins has been facilitated by the use of test fungi. The use of a biotin-deficient yeast enabled Kogl and Tonnis (1936) to isolate biotin for the first time as a pure compound. GENERAL PROCEDURES The following discussion of the steps involved in microbiological assay may serve also as a guide to the quantitative study of the physiology of fungi. Such studies are the surest way to gain knowledge and under- standing of the physiology of the fungi. Selection of test organisms. The first requirement of a test organism is specificity for the compound under assay. A fungus which responds to either or both moieties of thiamine is less suitable than one which requires the intact thiamine molecule. Other considerations may out- weigh the advantages of strict specificity, but the response of the test organism to moieties, vitamers, and related compounds must be known. Other considerations besides specificity enter into the selection of test organisms. Test organisms should be easily maintained in culture, easily handled in the laboratory, and have stable biochemical character- istics. Rapid and uniform growth is desirable. The habit of growth is important. A fungus which forms mucilaginous colonies which adhere to the walls of the flasks is difficult to harvest, and yeasts which clump are difficult to determine by turbidimetric methods. The basal medium. Except for the compound or element under investigation the basal medium should be complete and balanced. If a test organism is deficient for more than one factor, all the factors except the one under investigation should be present in optimum amounts. Other requirements are easily available sources of carbon and nitrogen and a medium which is adequately buffered in the optimum pH range. 210 PHYSIOLOGY OF THE FUNGI The basal medium should be essentially free from the vitamin or other factor under test. The response of the test organism to the basal medium should be slight; this value is known as the blank, or control. The size of the blank depends upon the residual concentration of the factor in the basal medium and the amount and kind of inoculum used. The degree to which a basal medium should be freed of the substance under test depends upon the sensitivity of the test organism. The best basal medium for any test organism can be determined only after a prolonged investigation of the nutritional requirements of the organism. This arduous task is too infrequently attempted. Fre- quently, it is desirable to use some natural material in the medium. A complex medium which supplies several sources of carbon and nitrogen as well as other organic compounds may support more rapid growth than a simple minimal medium. The sample being analyzed may contain compounds which stimulate or depress growth. Stimulation of growth due to the presence of acces- sory factors, is perhaps more often encountered than growth depression. The adequacy of the basal medium may be tested by comparing the growth curve obtained on the sample with the standard curve. If the response of the test organism to the sample is due solely to the factor con- tained in the sample, the two curves will be identical. The presence of inhibiting substances in the sample is detected when the sample curve falls below the standard curve. Stimulating substances are revealed by an upward drift of the sample curve. If biologically pure compounds were available, the preparation of basal media for assay purposes would be greatly simplified. No general method of purification is useful for all purposes. Riboflavin is destroyed by light, and media can be freed of this vitamin by exposure to strong illumination. Activated charcoal (Norit or Darco) is very useful in adsorbing residual traces of many vitamins. Recrystallization of sugars, asparagine, and mineral salts is helpful in some instances. Casein is extracted with hot alcohol to remove vitamins. The essential micro elements may be removed in the ways discussed in Chap. 5. Frequently reagents made by one manufacturer are purer in certain respects than those of another. Three basal media which have been used for fungi in microbiological assays are given below. Glucose-Asparagine Glucose 30 e, Asparagine 1 g. MgS04-7HoO 0.5 g. KH2PO4 1.5 g. Distilled water to make 1 liter FUNGI AS TEST ORGANISMS 211 This medium was used for thiamine assay using Phycomyces blake- sleeanus as the test fungus (Schopfer, 1945). Sucrose-Ammonium Tartrate-Ammonium Nitrate Sucrose 20 g. KH2PO4 1 g. MgS04-7H20 0.5 g. Ammonium tartrate 5 . 0 g. NH4NO3 1.0 g. NaCl 0.1 g. CaCli 0.1 g. B 0.01 mg. Mo 0 . 02 mg. Fe 0.2 mg., Cu 0.1 mg. Mn 0.02 mg. Zn 2.0 mg. Bio tin 5 Mg Distilled water to make 1,000 ml. This medium was used by Horowitz and Beadle (1943) and by Beadle (1944) for the assay of choline and inositol by biochemical mutants of Neurospora crassa. Glucose-Casein Hydrolysate Glucose 25 g. Casein hydrolysate equivalent to 2 g. casein MgS04-7H20 0.5 g. KH2PO4 l.Og. Fumaric acid 1 . 32 g. NaaCOs 1.12 g. Fe+ + + as sulfate 0.2 mg. Zn+ + as sulfate 0.2 mg. Mn+ + as sulfate 0.1 mg. Distilled water to make 1 liter This medium was used by Leonian and Lilly (1945) for the assay of certain vitamins. Various deficient yeasts and filamentous fungi were used as test organisms. This medium is suitable for testing fungi for vitamin deficiencies. Preparing for an assay. In general, the compound being assayed should be brought into aqueous solution before assaying. Many vita- mins occur in a ''bound" condition and must be liberated before analysis. The procedure used to liberate bound vitamins depends upon the vita- min involved, as well as the nature of the substance being assayed. Snell (1948) has listed tentative methods for the liberation of the various vitamins. In general, acid or enzymatic hydrolysis is used. Proteins are hydrolyzed before amino-acid assay. Acid hydrolysis is destructive 212 PHYSIOLOGY OF THE FUNGI to certain amino acids, especially tryptophane. Alkaline hydrolysis of proteins has been recommended for this amino acid (Greene and Black, 1944). The concentrations of the standard compound and of the sample for assay should be so chosen that the response of the test organism is roughly linear. Every concentration should be run in duplicate. Control flasks to which neither the standard compound nor the assay sample have been added should form a part of every assay. This provides a means of evaluating the basal medium and should never be omitted. The type of culture vessel and the volume of the basal medium used will depend upon the test organism. Bacteria are frequently cultured in test tubes. These are also useful for yeasts. Uniform test tubes which can be used in a photoelectric colorimeter allow measurement of turbidity without transfer (Lindegren and Raut, 1947). The filamentous fungi are usually cultured in Erlenmeyer flasks. The volume of medium should be so chosen that the liquid is less than 1 cm. deep. All glassware must be clean. Accuracy in measuring the basal medium and the known and unknown solutions is essential. Inoculation and incubation. The medium upon which the inoculum is grown should be complete and contain an adequate but not excessive amount of the factor under investigation. Certain fungi, especially the yeasts, cease to be deficient for certain vitamins when continuously cul- tured upon media free from these factors. Spore inoculum may be used with advantage with many filamentous fungi. Frequently it is desirable to use germinated spores for inoculum. Phycomyces blakesleeanus spores require the Z factors for rapid germina- tion (Robbins, 1940). If the test sample contains these factors and the basal medium does not, early growth will be more rapid in the sample series. It is convenient to germinate the spores of this fungus and others by preparing a spore suspension in dilute peptone solution a few hours before inoculation. These germinated spores grow essentially without interruption and shorten the time of incubation. Fragmented mycelium may also be used to advantage. A uniform amount of inoculum must be used. This is easy to achieve when a suspension of spores or fragmented mycelium is used. Inocula of these types provide a multitude of growing points, which results in uniform growth. Disks of mycelium on agar are, in general, unsatisfactory. An obvious advantage of using a large amount of inoculum is the shorter time required for an assay. However, there is danger of intro- ducing with a large inoculum enough of the substance under investigation to give abnormally high blanks. Washing the inoculum with sterile distilled water reduces this hazard but increases the work and multiplies the chances of contamination. A very small inoculum results in a longer lag period, and the time required for analysis may be prolonged. FUNGI AS TEST ORGANISMS 213 Test organisms during an assay should be cultured under uniform conditions with respect to light and temperature. In general, the fila- mentous fungi should not be agitated during the period of incubation. Yeasts are frequently grown with continuous or intermittent shaking. There are two schools of thought concerning the time of incubation for assay. The first recommends a uniform short period of growth and determination of the yield before the organism reaches its maximum development. There is a saving in time in this method, but the influence of accessory factors in the sample may make such results unreliable. A comparison should always be made between the analytical data for a short and a long period of incubation before choosing the length of incu- bation period. In general, we feel that assays tend to be more reliable when the period of incubation is long enough to allow maximum development of the test organism. Measuring the response. The methods used for measuring the response of test organisms vary. The growth response of bacteria may be measured either by titrating the acid produced or by determining the turbidity with a suitable photoelectric colorimeter. The growth response of yeasts may be measured as turbidity, or the cells may be weighed. The first procedure is by far the simpler. The growth of filamentous fungi is commonly measured by collecting the mycelium and determining the drj^ weight (see discussion in Chap. 3). Calculation of results. A growth curve (acidity, turbidity, or weight) is plotted from the response of the test organism to the different concen- trations of the standard substance. The concentration of the substance in the sample is then calculated from the standard curve. It is necessary to use a new standard curve for each series of assays. Unsuspected variations in the basal medium and in technique from day to day make this precaution necessary. In making the calculations, it is assumed that equal amounts of the substance, whether as a pure compound or in the sample, will cause the same amount of response by the test organism. It is customary to report the concentrations of vitamins and micro essen- tial elements in micrograms per gram of original sample. As an example of the type of calculation involved in an assay, the standard curve (Fig. 43) and protocol of a biotin assay are given below. The substance assayed was air-dry yeast cells. Biotin was liberated from the sample by acid hydrolysis, and the cell extract was neutralized and made up to such volume that 1 ml. of hydrolysate was equivalent to 50 mg. of original yeast cells. The test organism, Saccharomyces cerevisiae, Gebriide Mayer strain, was incubated for 72 hr at 25°C. Twenty-five milliliters of glucose-casein hydrolysate medium was used per 250-ml. flask. The cultures were agitated 10 min. every hour. The data for the response of the test organism to varying amounts of yeast hydrolysate are given in Table 35. 214 PHYSIOLOGY OF THE FUNGI Table 35. Yield of Saccharomyces cerevisiae Cells Produced when Different Amounts of Yeast Hydrolysate Were Added to 25 Milliliters of a Biotin-free Glucose-Casein Hydrolysate Medium Yeast hydrolysate, Equivalent weight of sample, mg. Yield, mg. ml. per flask Flask 1 Flask 2 0.03125 0.0625 0.125 0.25 1.5625 3.125 6.25 12.5 9.2 20.2 32.8 48.0 8.9 19.0 32.8 47.6 The amount of biotin in the original sample may then be calculated. The amount of biotin in 6.25 mg. of the sample produced 32.8 mg. of 60 50 «40 S30 ©20 10 ^'■^'"'^ 1 ^( ^ / ^ c / i / / 0 0.001 0002 O004 0.006 0.008 Micrograms of biotin per flosk Fig. 43. Standard curve for a biotin assay using Saccharomyces cerevisiae, Gebriide Mayer strain, as the test fungus. Basal medium was glucose-casein hydrolysate, 25 ml. per 250-ml. Erlenmeyer flask. Cultures were incubated at 25°C., agitated 10 min. each hour, and harvested after 72 hr. dry yeast cells. From the standard curve this is seen to be equivalent to 0.0025 jug of biotin. The biotin content of the sample is therefore equal to 0.0025 X 1,000/6.25, or 0.4 /xg of biotin per gram of sample. VITAMIN ASSAYS It is beyond the intent of this chapter to include detailed information about techniques in connection with individual assays. The following references are useful for entry into the hterature. Schopfer (1945) has considered the philosophy underlying the use of microorganisms for assay. Leonian and Lilly (1945) investigated the use of many test organisms to assay the vitamin content of a single substance. This work showed that widely different assay values for some vitamins are obtained FVNGI AS TEST ORGANISMS 215 when different test organisms are used. The review of Snell (1948) represents the critical judgment of an active investigator in this field. While the filamentous fungi are frequently passed over in favor of bacteria and yeasts, they offer certain advantages when only simple apparatus is available, or where occasional assays are to be made. The test organisms for the specific vitamins listed below are in part those recommended by Snell (1948). Thiamine. Phycomyces hlakesleeanus. This fungus responds to the two moities of thiamine. Schopfer (1935, 1945) used a glucose-asparagine medium and used dry weight of mycelium to measure growth. This is an excellent organism to use in gaining experience with a microbiological assay. Schultz et al. (1942) used Saccharomyces cerevisiae (Fleischmann's baker's yeast) and measured the evolution of carbon dioxide, which was proportional to the thiamine content of the sample. Pyridoxine. Saccharomyces carlsbergensis. Snell (1945a) found that this yeast responds about equally to pyridoxine, pyridoxal, and pyridoxa- mine. Growth may be measured turbidimetrically or by weighing the cells. Differential assays for these three vitamers have been devised. p-Aminobenzoic acid. Neurospora crassa mutant. Various labora- tories have used this organism (Tatum et al, 1946). For the effect of pH on utilization of this vitamin see Wyss et al. (1944). Pantothenic acid. Saccharomyces carlsbergensis. Most, if not all, yeasts respond to the )3-alanine moiety of pantothenic acid. Atkin et al. (1944) noted that the incorporation of Z-asparagine in the basal medium reduced interference due to /3-alanine. Nicotinic acid. Lactobacillus arabinosus. This organism responds equally to nicotinic acid and nicotinamide. Growth may be measured either by titrating the acid produced, or turbidimetrically (Krehl et al., 1943). Zygosaccharomyces marxianus was used by Leonian and Lilly (1945). Inositol. Neurospora crassa mutant. This mutant was first used by Beadle (1944) to assay inositol. It is an easy organism to handle, and since this mutant forms few conidia, it is not a great source of contamina- tion to a laboratoiy. Snell (1948) recommends the use of Saccharomyces carlsbergensis for inositol assay. Biotin. Saccharomyces cerevisiae. Various strains have been used. Many, if not all, strains respond also to desthiobiotin (Lilly and Leonian, 1944). The existence of many biotin vitamers makes the choice of a test organism difficult. Neurospora crassa and N. sitophila may also be used. It is probable that some of the divergence of assay values obtained when different test organisms are used is due to biotin complexes. Such a complex, biocytin, has been isolated by Wright et al. (1950). The analytical results were unchanged by acid hydrolysis when Lactobacillus 216 PHYSIOLOGY OF THE FUNGI casei was used but were increased when L. arahinosus was the test organism. Riboflavin. Lactohacillus casei. Fatty acids stimulate growth. Growth may be measured by titrating the acid formed, or turbidimetri- cally (Roberts and Snell, 1946). It is probable that mutants of Neuro- spora deficient for this vitamin may also be used in assay. AMINO-ACID ASSAYS The importance of the amino-acid composition of proteins used in animal nutrition makes any advance in analytical methods of great interest and value. The general techniques for amino-acid determina- tions by microbiological procedures are the same as for other assays. The first requirement of this type of microbiological assay is a suitable test organism. Few fungi isolated from nature are deficient for amino acids. For this reason bacteria have been extensively used. The follow- ing references will give an entry into the literature on the use of bacteria for amino-acid assay: Hutchings and Peterson (1943); Shankman (1943); Dunn et at. (1944); Snell (1945); and Horn et at. (1950). Some mutants of N'eurospora have been found to be deficient for amino acids. Mutants having the following amino-acid deficiencies have been studied: leucine, isoleucine, valine, lysine, methionine, serine, or glycine. Only the mutant deficient for leucine appears to have been much used in microbiological assay (Ryan and Brand, 1944; Brand et al., 1945). The growth of a lysine-deficient mutant was completely inhibited by arginine when the molecular ratio of arginine to lysine was 2 to 1 (Doermann, 1944). Ryan (1948) has considered the possibility of microbiological assay of amino acids by observing the percentage of germination of conidia from deficient mutants in the presence of different concentrations of the specific amino acid. An assay can be completed within a few hours by this method. Unfortunately the inhibiting action of certain amino acids introduces complications into the proposed method. Mutations of Neurospora and certain other fungi have been induced by chemicals, such as nitrite or nitrous acid, colchicine, nitrogen mustard gas, and hydrogen peroxide, or by irradiation with ultraviolet and X rays. These mutants are frequently characterized by inability to synthesize various metabolites. They differ from the parent wild type in that one or more genes have been inactivated. It is thought that each gene con- trols a single biochemical reaction. Mutants having the same gross deficiency may differ in the specific gene inactivated. Horowitz (1947) studied four mutants of Neurospora which were unable to synthesize methionine from inorganic sources of nitrogen and sulfur. One of these mutants was able to grow in the presence of cysteine, cysta- FUNGI AS TEST ORGANISMS 217 thionine, homocysteine, and methionine. The second was unable to utihze cysteine but was able to utilize the other three compounds. The third isolate utilized either homocysteine or methionine, while the fourth isolate utilized only methionine. From these results the steps in the synthesis of methionine and the genes inactivated may be summarized as gene 4 gene 3 gene 2 gene 1 follows : > cysteine > cystathionine > homocysteine — -^ methi- onine. From similar studies Srb and Horowitz (1944) concluded that Neurospora synthesizes arginine as follows: ornithine — -^ citrulline > arginine. Fungus mutants have proved to be powerful tools for investigating pathways of synthesis and utilization of vitamins, amino acids, and other compounds, and in studies of biochemical mutations. From these studies also comes the realization that each step in the synthesis or utilization of a compound may be controlled or limited by a specific gene. The review papers of Bonner (194G) and Beadle (1945, 1945a) should be consulted for further information and literature citations. ASSAYS FOR ESSENTIAL ELEMENTS Microorganisms may be used to determine the presence of essential elements. In view of the speed and accuracy of chemical and spectro- scopic methods, it might be assumed that microorganisms would be of little value in such applications. The value of microbiological tests would appear to be in applications where availability as well as total amounts are of importance. Problems of this sort frequently arise in connection with mineral deficiencies in soil. It is recognized that the absolute content of an essential element in a soil may not measure the availability of that element for green plants. Microbiological and chem- ical methods of analysis must be correlated with plant tests before they are of much value. The possible number of test organisms is unlimited except for the important considerations of sensitivity, ease of handling, and time required to make an assay. In practice, only a few organisms have been used. There exists a wide field for investigations dealing with the cor- relation between availability to microorganisms and availability to green plants of certain essential elements in soil. Copper. Mulder (1939-1940) used Aspergillus niger to determine copper in soil. The range of concentrations in the standard series was 0.0 to 2.5 Mg Cu++ per culture; 40 ml. of medium was used in liter flasks. One gram of sterile soil was used as the sample. The method of measur- ing the response of A. niger to copper was very simple, inasmuch as the number and color of the spores produced were functions of the copper content of the medium. No spores developed on the control medium, but 218 PHYSIOLOGY OF THE FUNGI with increasing concentrations of copper the spores were yellow, yellow- brown, gray-brown, brown, and black. The color of the spores produced on copper-deficient media by different isolates of A. niger varied. Excellent correlation between the copper content of various soils as determined by this method and the incidence of copper deficiency in grain was found. Some of Mulder's results are presented in Table 36. Table 3G. The Correlation of Copper Deficiency in White Oats and the Copper Content of the Soil as Determined by Aspergillus niger Method All soil was from the same field. (Mulder, Antonie van Leeuwenhock 6, 1940.) Available Copper, Condition of Oats Mg per G. of Soil Severely diseased 0 . 25 Less severely diseased 0.8 Healthy (from a portion of the field not showing the disease) 1.7 Healthy (copper sulfate added to the soil) 2.5 Magnesium. Smit and Mulder (1942) postulated that a microbio- logical method would show better correlation with magnesium deficiency in green plants than chemical methods. This was confirmed for the Netherlands soils investigated. Azotobacter chroococcum and Aspergillus niger were used as test organisms. Preference w^as given to the fungus inasmuch as only 4 to 5 days w-ere required for an assay. A simple technique was used, and visual comparison w^as sufficiently accurate to diagnose magnesium deficiency in soils. Potassium. Aspergillus niger was used by Niklas and Toursel (1940) to determine available potassium and other elements in soils. These authors weighed the mycelium produced. Rogosa (1944) has shown that Lactobacillus casei may be used to determine small amounts of potassium. rABLE 37. The Effect of Molybdenum Content of a Glucose-Nitrate Medium UPON Yield of Mycelium and Sporulation of Aspergillus niger (Mulder, Plant and Soil 1, 1948.) Mg Na2Mo04-2H.A in 50 ml. medium Mg. mycelium per culture Sporulation Appearance of mycelium 0.0 0.0025 0.010 0.050 165 294 558 868 0 0 0 Normal Entirely mucous Partially mucous Partially mucous Normal Molybdenum. The amount of this element needed by fungi and green plants is greater when nitrogen is supplied as nitrate than when ammo- nium nitrogen is furnished. This fact introduces a complication into the microbiological assaj^ of molybdenum in that the sample must be ashed FUNGI AS TEST ORGANISMS 219 before analysis. It is probable that amino acids and other nitrogen sources containing reduced nitrogen would also affect the amount of molybdenum needed. Mulder (1948) investigated the use of Aspergillus niger as a test organism (Table 37). For further discussion and refer- ences to the use of microorganisms in essential-element assay see Vande- caveye (1948). SUGARS Yeasts and other microorganisms have been used to separate optical isomers and complex mixtures of sugars. Pasteur (18G0) used Pemcillium glaucum to obtain the "unnatural" isomer of tartaric acid from rfZ-tartaric acid. Fischer and Hertz (1892) used brewer's yeast to ferment D-galac- tose, while L-galactose in the same medium was not utilized. Auernheimer el al. (1948) used the specific fermentative powers of Hansenula suaveolens and Candida guilliermondi in the separation of L-arabinose and D-xylose obtained from the hydrolysis of straw and corn cobs. H. suaveolens does not utilize L-arabinose, while C. guilliermondi utilizes both pentoses. Saccharomyces carlshergensis was used to demonstrate the absence of D-glucose in the hydrolysates. These yeasts were used in conjunction with chemical methods of analysis. Appling et al. (1947) found Sac- charomyces carlshergensis var. mandschuricus to ferment D-galactose but not L-galactose. Similarly, H. suaveolens utilized D-xylose but not L-xylose. These citations indicate the usefulness of yeasts and other organisms in the solution of problems difficult to solve by other methods. The value of microorganisms in such applications is due to their specificity. TESTS FOR CERTAIN METABOLIC PRODUCTS Fungi excrete into the media in w^hich they grow various physiologically active substances. In the older literature these are referred to as staling 'products. Among the metabolic products are those which may stimulate or inhibit growth and reproduction. The kind and the amount of com- pounds excreted depend upon the particular fungus involved as well as the composition of the medium. The effect of the metabolic products ot one fungus upon another is simply demonstrated when fungi are gro^vn in association. The beneficial effect of one fungus upon another was demonstrated by Kogl and Fries (1937). Neither Nematospora gossypii or Polyporus adustus grew when inoculated alone into a synthetic medium, but when both fungi were inoculated together in the same flask, both began to grow rapidly after about a week. N. gossypii is deficient for biotin but synthesizes thiamine, while P. adustus is deficient for thiamine but synthesizes biotin. Kogl and Fries called this artificial symbiosis. Schopfer and Guilloud (1945) cite other examples in connection with work on strains of Candida guilliermondi involving vitamin deficiencies. 220 PHYSIOLOGY OF THE FUNGI By using a series of test organisms of known deficiencies, it is easy to demonstrate that fungi excrete vitamins. It is a common experience to find deficient fungi growing in association with contaminants. The method is simple and consists of inoculating plates of vitamin-free medium with two test fungi (Fig. 44). Not all fungi excrete the same amount of a Fig. 44. Test demonstrating the excretion of biotin by Aspergillus 7'ugulosus (right), whengrownwith Sordariafimicola (biotin-deficient) on vitamin-free glucose-asparagine medium. Sordaria (left) made only slight growth until it approached the colony of Aspergillus, where a zone of stimulated growth is evident. given vitamin. This may be shown by choosing test fungi such as Sordaria fimicola, which requires more biotin for fruiting than for growth. Some fungi excrete enough biotin to allow grow^th of S. fimicola, while others excrete enough biotin to allow reproduction also. Other com- pounds besides the vitamins may be excreted and favor the growth of other organisms. Further instances of the favorable effect of one fungus on the sporulation of another are discussed in Chap. 14. The metabolic products of one fungus may inhibit the growth of another. This phenomenon may be frequently observed on contaminated plates (Fig. 45). Fleming (1929) discovered the action of penicillin in this way. Many fungi apparently produce substances which inhibit the germina- tion of their spores. Schopfer (1933) found that spores of Phy corny ces blakesleeanus would not germinate on agar media upon which this fungus had grown. If such a "staled" plate was autoclaved, the medium would then allow germination and growth of P. blakesleeanus. These results FUNGI AS TEST ORGANISMS 221 indicate that the spore-inhibiting substance was either volatile or unstable. This inhibitory substance was not identified. Fig. 45. Test for antibiotic production by growing two organisms in association on the same agar plate. Helminthosporium sativum on the left and an unidentified actinomycete on the right. TESTING FABRIC PROTECTANTS While the deterioration of cellulosic materials exposed to the weather or in contact with the soil is not solely due to the action of bacteria and fungi, these organisms are the chief agents of destruction. The problem of deterioration of cellulosic materials has received a vast amount of attention, especially in connection with military materiel in humid tropic climates. Work on this problem involves the identification of the respon- sible microorganisms, laboratory tests, and use of test fungi in evaluating protectants. The basis of the various methods for determining cellulolytic activity consists in inoculating cotton duck or other test material with the fungi under test. The degree of cellulolytic activity is determined by measur- ing the decrease in tensile strength of the test specimen. The test medium used is usually an inorganic salt solution having pH 6.8. It is desirable to use a buffered medium inasmuch as cellulase is most active around pH 7. White et at. (1948) note that many fungi which are strongly cellulolytic under laboratory conditions cause but httle damage in the field. They believe that, under a given set of natural environ- mental conditions, the actual decay of fibers is caused by a relatively few species of fungi. Among the strongly cellulolytic fungi are Mem- 222 PHYSIOLOGY OF THE FUNGI noniella echinata (the variability in strains in laboratory tests is possibly correlated with biotin deficiency), Chaetomium spp., especially C. glo- bosian (Greathouse and Ames, 1945), Myrothecium verrucaria (as strong a cellulose decomposer as yet found in laboratory tests), Trichoderma viride, and Thielavia sepedonium. The reduction in tensile strength of cotton duck maintained under specified conditions is used as a measure of the destructive effects of fungi on fabrics. The data in Table 38 are taken from White et al. (1948). Table 38. Assay of Fungi for Cellulolytic Activity Based upon Loss of Tensile Strength of Cotton Duck (White et al, Mrjcologia 40, 1948.) Species Aspergillus niger PQMD 25a A. terreus PQMD 72f Chaetomium funicolum PQMD 351. C. globosum PQMD 32b Fusarium oxysponim Fla C-8 Gliomastix convoluta PQMD 4c Myrothecium verrucaria PQMD 70h Thielavia sepedonium PQMD 47g. . Trichoderma viride PQMD 6a T. viride PQMD 63d Strength retained, % 6 days 9 days 12 days 100 103 105 67 0 18 42 32 0 — 49 36 30 0 15 51 22 0 — 18 10 8 100 99 98 Growth at end of experiment 2 4 4 4 3 4 4 4 4 0 The evaluation of protective fungicides for fabrics, paper, and other cellulosic materials consists in comparing the effects of known cellulolytic fungi upon treated and untreated specimens of material. In addition to causing loss of tensile strength, some fungi cause great damage by surface growth (mildew). Abrams (1948) has reviewed the techniques used at the Bureau of Standards for testing mildew- and rotproofing agents. Aspergillus niger was used to determine mildew resistance, and the effectiveness of various treatments was evaluated by visual observa- tion. Chaetomium globosum and a species of Penicillium (USDA 66) were used in rot-resistance tests. Of some 36 compounds tested, copper naphthenate and pyridyl mercury compounds were most effective. The effectiveness of the fungicides varies with the test organisms used. For data on fungicide evaluation the reader is referred to Abrams (1948). SUMMARY The use of microorganisms for analytical purposes is based upon specific biochemical characteristics of selected test organisms. Within a certain range of concentration, the response is proportional to the amount of FUNGI AS TEST ORGANISMS 223 test substance present in the medium. Among the substances for which quantitative assay procedures have been developed are the vitamins, amino acids, and essential elements. Microorganisms have also been used to discover pathways of biochemical synthesis and degradation, to separate isomers, and for other analytical purposes. The essential features of a microbiological assay are (1) a suitable test organism, (2) a suitable basal medium essentially free from the substance under test, (3) preparation of the sample, (4) a reference standard (a pure compound where possible), (5) two series of cultures to which a known range of concentrations of the standard and unknown have been added, (6) uniform inoculation, (7) incubation under uniform conditions, (8) measuring the response of the test organisms, (9) construction of a standard curve, and (10) calculating the results. When microbiological assay procedures are used, it is unnecessary to isolate the compound being assayed from the other constituents present in the sample. The preparation of the sample for assay is usually simple and ordinarily involves hydrolysis. Microbiological procedures usually require a short time to complete. The amount of sample needed is small, which is an important consideration in some problems. Microbiological assays are invaluable, provided that suitable test organisms are available, in devising chemical procedures for the isolation of new vitamins and other physiologically active compounds. Biochemical mutants of Neurospora and other fungi are particularly useful in determining the pathways of synthesis of amino acids and other compounds. REFERENCES Abrams, E.: Microbiological deterioration of organic materials, its prevention and methods of test, Natl. Bur. Standards (U.S.), Misc. Pub. 188, 1948. Appling, J. W., E. K. Ratcliff, and L. E. Wise: Chemical and microbiological differentiation of enantiomorphs of galactose and xylose, Anal. Chem. 19 : 496- 497, 1947. Atkin, L., W. L. Williams, A. S. Schultz, and C. N. Frey: Yeast microbiological methods for determination of vitamins. Pantothenic acid, Ind. Eng. Chem., Anal. Ed. 16: 67-71, 1944. AuERNHEiMER, A. H., L. J. WicKERHAM, and L. E. Schniepp: Quantitative deter- mination of hemicellulose constituents by fermentation, Anal. Chem. 20: 876-877, 1948. Beadle, G. W. : An inositoUess mutant strain of Neurospora and its use in bioassays, Joiir. Biol. Chem. 156 : 683-689, 1944. Beadle, G. W. : Biochemical genetics, Chem. Revs. 37 : 15-96, 1945. Beadle, G. W. : Genetics and metabolism in Neurospora, Physiol. Revs. 25 : 643-663, 1945a. Bonner, D.: Biochemical mutations in Neurospora, Cold Spring Harbor Symposia Quant. Biol. 11 : 14-24, 1946. Brand, E., F. J. Ryan, and E. M. Diskant: Leucine content of proteins and food- stuffs, Jour. Am. Chem. Soc. 67: 1532-1534, 1945. 224 PHYSIOLOGY OF THE FUNGI DoERMANN, A. II.: A lysineless mutant of Neurospora and its inhibition by arginine, Arch. Biochem. 5 : 373-384, 1944. Dunn, M. S., S. Shankman, M. N. Camien, W. Frankland, and L. B. Rockland: Investigations of amino acids, peptides, and proteins. XVIII. The amino acid requirements of Leuconostoc viesenteroides P-60, Jour. Biol. Chem. 156: 703-713, 1944. FxscHER, E., and J. Hertz: Reduction der Schleimsaure, Ber. d. deut. chem. Ges. 25: 1247-12(51, 1892. ■^Fleming, A.: On the antibacterial action of cultures of a Penicillium, with special reference to their use in the isolation of B. influenzae, Brit. Jour. Exptl. Path. 10: 226-236, 1929. Greathouse, G. a., and L. M. Ames: Fabric deterioration by thirteen described and three new species of Chaetomium, Mycologia 37 : 138-155, 1945. Greene, R. D., and A. Black: The microbiological assay of trytophane in proteins and foods. Jour. Biol. Chem. 155 : 1-8, 1944. Horn, M. J., D. B. Jones, and A. E. Blum: Methods for microbiological and chemi- cal determinations of essential amino acids in proteins and foods, f7.»S. De-pt. Agr. Misc. Pub. 696, 1950. *HoRowiTZ, N. H.: Methionine synthesis in Neurospora. The isolation of cysta- thionine. Jour. Biol. Chem. 171 : 255-264, 1947. Horowitz, N. H., and G. W. Beadle : A microbiological method for the determina- tion of choline by use of a mutant of Neurospora, Jour. Biol. Chem. 150 : 325-333, 1943. Horsfall, J. G.: Fungicides and Their Action, Chronica Botanica Co., Waltham, 1945. HuTCHiNGS, B. L., and W. H. Peterson: Amino acid requirements of Lactobacillus casei, Proc. Soc. Exptl. Biol. Med. 52 : 36-38, 1943. Kogl, F., and N. Fries: Ueber den Einfluss von Biotin, Aneurin und Meso-Inosit auf das Wachstum verscheidener Pilzarten, Zeit. physiol. Chem. 249: 93-110, 1937. KoGii, F., and B. Tonnis: Ueber das Bios-Problem. Darstellung von krystallisier- tem Biotin aus Eigelb, Zeit. physiol. Chem. 242 : 43-73, 1936. Krehl, W. a., F. M. Strong, and C. A. Elvehjem: Determination of nicotinic acid. Modifications in the microbiological method, Ind. Eng. Chem., Anal. Ed., 15: 471-475, 1943. Leonian, L. H., and V. G. Lilly: The comparative value of different test organisms in the microbiological assay of B vitamins. West Va. Agr. Expt. Sta. Bull. 319, 1945. Lilly, V. G., and L. H. Leonian: The anti-biotin effect of desthiobiotin, Science 99 : 205-206, 1944. LiNDEGREN, C. C, and C. Raut: The effect of the medium on apparent vitamin- synthesizing deficiencies of microorganisms, Ann. Missouri Botan. Garden 34: 75-84, 1947. Mulder, E. G.: On the use of micro-organisms in measuring a deficiency of copper, magnesium and molybdenum in soils. Anionic van Leeuwenhoek 6: 99-109, 1939-1940. Mulder, E. G. : Importance of molybdenum in the nitrogen metabolism of micro- organisms and higher plants, Plant and Soil 1 : 94-1 19, 1948. NiKLAS, H., and O. Toursel: Die Bodenuntersuchung mittels Aspergillus niger, Bodenkunde und Pflxinzen erndkr. 18 : 79-107, 1940. Pasteur, L. : Note relative au Penicillium, glaucum et a la dissymetrie moleculaire des produits organiques naturels, Compt. rend. acad. set. 61 : 298-299, 1860. FUNGI AS TEST ORGANISMS 225 RoBBiNS, W. J.: Effect of extracts of Phycomyces upon its development, Am. Jour. Botany 27: 559-564, 1940. Roberts, E. C, and E. E. Snell: An improved medium for microbiological assays with Lactobacillus casei, Jour. Biol. Chem. 163 : 499-509, 1946. RoGOSA, M.: Microbiological method for the quantitative determination of small quantities of potassium, Jour. Biol. Chem. 154: 307-308, 1944. *Ryan, F. J. : The germination of conidia from biochemical mutants of Neurospora, Am. Jour. Botany 35: 497-503, 1948. Ryan, F. J., and E. Brand: A method for the determination of leucine in protein hydrolysates and in foodstuffs by the use of a Neurospora mutant, Jour. Biol. Chem. 154: 161-175, 1944. ScHOPFER, W. H.: Etude d'un cas de stimulation unilaterale et d'un cas d'inhibition chez un micro-organisme, Compt. rend. soc. phys. hist. nat. Genhve. 50: 152-154, 1933. ScHOPFER, W, H.: Recherches sur I'emploi possible d'un test vegetal pour la vitamine Bi. Essai d'etalonnage. Bull. soc. chim. biol. 17: 1097-1109, 1935. ScHOPFER, W. H. : Les Tests microbiologiques pour la determination des vitamines, Experientia 1 : 1-68, 1945. ScHOPFER, W. H., and M. Guilloud: Un cas de stimulation unilateral chez las microorganismes explique par une action vitaminique, Verhandl. naturforsch. Ges. Basel 61: 299-314, 1945. ScHULTZ, A. S., L. Atkin, and C. N. Frey: Determination of vitamin Bi by yeast fermentation method. Improvements related to the use of sulfite cleavage and a new fermentometer, Ind. Eng. Chem., Anal. Ed. 14: 35-39, 1942. Shankman, S.: Amino acid nutrition of Lactobacillus arabinosus, Jour. Biol. Chem. 150:305-310, 1943. Smit, J., and E. G. Mulder: Magnesium deficiency as the cause of injury in cereals, Mededeelingen van de Landbouwhoogeschool, Deel 46, Verhandeling 3, 1942. Snell, E. E. : The microbiological assay of amino acids. Advances in Protein Chem. 2 : 85-118, 1945. Snell, E. E. : The vitamin Be group. Evidence for the occurrence of pyridoxamine and pyridoxal in natural products, Jour. Biol. Chem. 157: 491-505, 1945a. *Snell, E. E.: Use of microorganisms for assay of vitamins, Physiol. Revs. 28: 255- 282, 1948. Srb, a. M., and N. H. Horowitz: The ornithine cycle in Neurospora and its genetic control, Jour. Biol. Chem. 154: 129-139, 1944. Tatum, E. L., M. G. Ritchey, E. V. Cowdry, and L. F. Wicks: Vitamin content of mouse epidermis during methylcholanthrene carcinogenesis. I. Biotin, choline, inositol, p-aminobenzoic acid and pyridoxine, Jour. Biol. Chem. 163 : 675-682, 1946. Vandeca\teye, S. C. : Biological methods of determining nutrients in soil in Diagnos- tic Techniques for Soils and Crops (edited by H. B. Kitchen), The American Potash Institute, Washington, 1948. * White, W. L., R. T. Darby, G. M. Stechert, and K. Sanderson: Assay of cellu- lolytic activity of molds isolated from fabrics and related items exposed in the tropics, Mycologia 40 : 34-84, 1948. Wright, L. D., E. L. Cresson, H. R. Skeggs, T. R. Wood, R. L. Peck, D. E. Wolf, and K. Folkers: Biocytin, a naturally-occuring complex of biotin. Jour. Am. Chem. Soc. 72: 1048, 1950. Wyss, O., V. G. Lilly, and L. H. Leonian: The effect of pH on the availability of p-aminobenzoic acid to Neurospora crassa, Science 99: 18-19, 1944. CHAPTER 11 METABOLITE ANTAGONISTS This chapter and the one following will deal with chemical compounds which inhibit, injure, or kill fungi. Much can be learned about "normal" physiological processes by studying the factors which interfere with them. The ideas to be discussed here are applicable to the entire field of phys- iology, and some of our illustrative material will deal with organisms other than fungi. The reviews of Woolley (1944), Welch (1945), Wright (1947), Mcllwain (1947), and Roblin (1946, 1949) are extensive and well documented and should be consulted for additional references. Metabolites are chemical substances which are essential for the func- tioning and maintenance of living cells. Metabolites may be synthesized by the organism or obtained from the medium, e.g., vitamins, amino acids, etc. An antimetabolite, or antagonist, is a compound which interferes with the utilization of a normal metabolite. Wright (1947) has classified antagonists (more specifically antivitamins) on the basis of their mode of action: (1) those which act by virtue of destroying or inactivating a metabolite; (2) those which combine irreversibly with enzymes (non- competitive inhibition) ; and (3) those which combine with enzymes but w^hich may be displaced by increased concentration of the normal metabo- lite (competitive inhibition). Noncompetitive enzyme inhibition is so called because an increase in the concentration of the normal coenzyme or metabolite molecules does not reverse the inhibition. Inhibitors of this type act by combining with some atom or molecular group of either a coenzyme or an apoenzyme. Among inhibitors of this type we may list the heavy metals, various organic mercury and arsenic compounds, iodoacetate, and quinones, which inactivate enzymes by combining with free sulfhydryl groups (see Singer, 1945, and McElroy, 1947, for references). Among the inhibitors which act on the iron-porphyrin enzymes are cyanide, azide, hydrogen sulfide, and carbon monoxide. Most of the discussion to follow will deal with competitive antagonists. Metabolite antagonists are analogues of normal metabolites, but not all analogues of a metabolite are necessarily antagonists. These "for- eign" molecules, because of their close resemblance to normal metabolites, combine with enzymes in the same manner as normal metabolites. How- 226 METABOLITE ANTAGONISTS 227 ever, these foreign molecules are not transformed by the enzyme to which they are bound. If the antagonist is an analogue of a coenzyme, it presumably forms a pseudoholoenzyme which is unable to function. The close structural relation between a metabolite (p-aminobenzoic acid) and its antagonist (sulfanilamide) is shown in Fig. 46. 6.7 A. H H N J A ! — ►|2.3A.|-^ p-Aminobenzoate Ion —►[2.4 A. Sulfanilamide Fig. 46. Interatomic distances and structural relationships of p-aminobenzoate ion and sulfanilamide. (Courtesy of Roblin, Chem. Eng. News 27 : 3624, 1949. Published by permission of American Chemical Society.) In spite of the large number of compounds which have been tested for antagonism, it is not possible to specify exactly what changes in metabolite molecules are required to produce antagonists. A single modification of a metabolite molecule is more hkely to produce an antagonist than two or more changes in structure. This is to be expected, for an antagonist must closely resemble the corresponding metabolite. Replacing a carboxyl group with a sulfonic-acid group has been effective in many instances. The specific action of enzymes has been likened to the relation of a lock and its key. Unless an enzyme and a substrate molecule are related in this fashion, no reaction will take place. A modern diagrammatic representation of the lock-and-key simile is shown in Fig. 47. The mechanism of competitive inhibition may be visuahzed by referring to this figure. Metabolite antagonists may be thought of as "wrong" keys, which jam the lock mechanism. As long as a false key is in the lock, it prevents the true key from entering and opening the lock. Compounds which resemble coenzymes in structure compete for the active surface of apoenzymes. Because of similarity in structure, an apoenzyme-foreign molecule complex, or pseudoholoenzyme is formed. Such a pseudoenzyme is unable to function. The reversal of enzyme inhibition in such instances is caused by the addition of more coenzyme molecules. The argument is the same when substrate analogues are involved. For example, 3-fluorophenylalanine inhibits the utihzation 228 PHYSIOLOGY OF THE FUNGI of phenylalanine (a normal metabolite) by Neurospora crassa (Mitchell and Neimann, 1947). The effect of an antagonist will depend upon the concentration of the normal metabolite present in the medium and cells and upon the organ- ism. In general, enzymes have a greater affinity for metabolites than for antimetabolites. Since both metabolite and antagonist compete for the same enzyme, the amount of inhibition will depend upon the relative concentrations rather than upon the absolute amounts of these compounds present. The amount of an inhibitor required to reduce the nAP Substrate + Products free enzyme Enzyme -substrate complex Fig. 47. A diagrammatic illustration of Fischer's simile that an enzyme and its substrate are related as are a lock and its key. (Courtesy of McElroy, Quart. Rev. Biol. 22 : 26, 1947. Published by permission of The Williams & Wilkins Company.) amount of growth to one-half will depend upon the ratio of inhibitor and metabolite present. In simple instances, at least, this ratio is equal to a constant and is called the inhibition constant, or index. The amounts of sulfadiazine and p-aminobenzoic acid required to reduce the amount of growth of Streptococcus faecalis R to one-half the normal value gave an inhibition index of 333 (Lampen and Jones, 1946). The inhibition index is valid only for the particular conditions used in an experiment and for the particular strains of the organism used. In the case of self-sufficient organisms the use of an amount of inhibitor less than that required for total inhibition will only decrease the rate of growth, and thus the inhibition index will change with the time of incuba- METABOLITE ANTAGONISTS 229 tion. This is due to the synthesis of the metabohte by the organism. Sulfanilamide inhibits the growth of Aspergillus niger, but the fun- gus overcomes this inhibition as the time of incubation is prolonged (Hartelius, 194G). The concentration of a metabolite in the control cultures should be less than the amount which allows maximum growth, because of the nonlinear response of an organism to the metabolite at high concentrations. The composition of the medium is an important consideration in any investigation of metabolite antagonism. If adequate amounts of a natural metabolite are present, the action of an inhibitor may be over- looked. Synthetic media should be used. The composition of the medium used may also affect the action of an inhibitor in another way. If metabolite A is transformed into metabolite B by an organism, the presence of metabolite B in sufficient amount for optimum growth may be expected to nullify any amount of an antagonist for metabolite A. An antagonist of metabolite B, however, would exhibit normal competitive inhibition, Shive and Macow (1946) have pointed out that, by the use of a suitable series of inhibitors, it is possible to follow the transformations of a given metabolite step by step. These authors designate this use of metabolite antagonists as inhibition analysis. Rydon (1948) found Bacterium typhosum to synthesize tiyptophane by the following steps: anthranilic acid — > indole — > tryptophane. The 2- and 4-methylanthran- ihc acids were potent inhibitors against anthranilic acid but not against indole or tryptophane. Certain analogues of indole and tryptophane were inhibitors of these metabolites. In discussing metabolite antagonists in a general way, it should be borne in mind that these compounds may inhibit only certain organisms, or a particular organism only under certain conditions. For example, desthiobiotin is a biotin antagonist for Ceratostomella pini and Lacto- hacillus casei, while this compound replaces biotin for many strains of Saccharomyces cerevisiae (Lilly and Leonian, 1944). Woolley (1944, 1946) is of the opinion that the established facts of inhibition and reversal are more important than the hypotheses which are adopted to explain these phenomena. However, the concept of competitive metabohte antagonism has been very useful in correlating a vast amount of experi- mental work in apparently unrelated fields. ANTIVITAMINS Antivitamins are known for all the water-soluble vitamins which have been synthesized and for at least one of the fat-soluble vitamins (vitamin K). p-Aminobenzoic acid antagonists. When the sulfonamides were intro- duced into medicine, it was quickly found that serum and other natural 230 PHYSIOLOGY OF THE FUNGI products antagonized the inhibitive action of sulfanilamide on the growth of certain bacteria. Rubbo and Gillespie (1940) discovered that p-amino- benzoic acid was a growth factor for certain bacteria. Woods (1940) found that p-aminobenzoic acid in low concentration overcame sulfanila- mide inhibition. A general theory was proposed by Fildes (1940) to explain the antagonism between metabolites and compounds having closely related structures. 1400 8 10 12 Doys of incubotion 16 18 20 Fig. 48. The effect of various concentrations of sulfanilamide (amounts per flask) upon the time of spore germination and upon the rate and amovnt of growth of Aspergillus niger in flasks containing 55 ml. of sucrose— ammonium sulfate medium at 32°C. (Drawn from the data of Hartelius, Compt. rend. trav. lab. Carlsberg, S^r. physiol. 24: 181, 1946.) Sulfanilamide was first considered to be antagonized by p-amino- benzoic acid, rather than the reverse. This was due to the discovery of the therapeutic value of sulfanilamide before it was known that p-aminobenzoic acid was a vitamin. The structural relation between these compounds has already been noted. The literature dealing with the sulfonamides is abundant, but most of it relates to bacteria and medicine. Relatively few papers have been published on the effects of these compounds on the growth of fungi. Hartelius (1946) investigated the effect of sulfanilamide upon the growth of Aspergillus niger and found that the amount of inhibition was dependent upon the amount of inoculum used, the concentration of sulfanilamide in the medium, and the time of incubation. The curves in Fig. 48 illustrate the effect of time of incubation on inhibition, a factor which is too often overlooked in experiments of this kind. The curves METABOLITE ANTAGONISTS 231 in Fig. 48 indicate that A. niger synthesizes either p-aminobenzoic acid or some other compound which reverses the inhibitory action of sulfa- nilamide. When p-aminobenzoic acid was added to the medium, sulfa- nilamide no longer inhibited the growth of yl. niger (Hartelius and Roholt, 1946). Other fungi have been shown to react like A. niger when cultured in media containing sulfanilamide (Fourneau et al., 1936). It has been assumed that self-sufficient fungi require the same vitamins as the deficient species. The synthesis of a vitamin may suggest its need but does not demonstrate it. Antivitamins (or other antimeta- bolites) provide a way of demonstrating the need of self-sufficient fungi for the vitamins they synthesize. Thus A. niger requires p-aminobenzoic acid just as Rhodotorula aurantica does, but this need can be demonstrated only in the presence of a specific reversible inhibitor such as sulfanilamide. This technique offers a possible way of discovering new vitamins and other metabolites. If a compound inhibits growth, it is worth while to search for compounds which overcome this inhibition reversibly. For most purposes sulfanilamide has been replaced by other sulfona- mides. However, sulfanilamide appears to be the most active sulfona- mide against fungi. For a review of the clinical aspects of the sulfona- mides in mycoses and for literature citations, see Wolf (1947). Stoddard (1947) has reported the sulfonamides to be of some value in controlling the X disease of peach (a virus). Addition of p-amino- benzoic acid lessened the effectiveness of the treatment. It is recognized that the simple Woods-Fildes theory of competitive inhibition is inadequate to explain completely the mechanism of sulfona- mide therapy. In vivo the environment is much more complex than in simple laboratory media. For further information and references to the literature, see Sevag et al. (1945) and Mudd (1945). Thiamine antagonists. Thiamine may be inactivated by an enzyme, thiaminase, which is found in fish viscera (Sealock et al., 1943) and prob- ably occurs in other organisms. Foxes which are fed raw fish may develop a thiamine-deficiency disease (Chastek paralysis). The mode of inactivation was further investigated by Krampitz and Woolley (1944), who found that thiamine was destroyed by a process of enzymatic hydrol- ysis whereby the thiazole and pyrimidine moieties were formed. Mucor ramanniamis (thiazole-deficient) and Endomyces vernalis (pyrimidine- deficient) were used as test organisms in the preliminary work. Another thiamine antagonist of unknown nature has been reported to occur in bracken fern (Weswig et al., 1946). Pyrithiamine, an analogue of thiamine, has been used in studies of competitive thiamine inhibition. Unfortunately, the exact structure of this compound is not known. In papers published before 1949 it was assumed that pyrithiamine had the structure now assigned to neopyri- 232 PHYHIOLOGY OF THE FUNGI thiamine (Wilson and Harris, 1949). Pyrithiamine appears to differ from neopyrithiamine in the amount of pyrimidine moiety it contains. The formula for neopyrithiamine is given below. N=C— NH2-HBr CH3 CH2— CH2— OH CHs — C C — CHj N— CH Br Neopyrithiamine Robbins (1941) found low concentrations of pyrithiamine to replace thiamine for Pythiomorpha gonapodyoides (pyrimidine-deficient), while high concentrations inhibited growth. Pyrithiamine did not replace thiamine for Phycomyces blakesleeanus (requires both moieties) or Phyto- phthora cinnamomi (requires intact thiamine). The inhibition of growth of various fungi and bacteria caused by pyrithiamine was overcome by increasing the thiamine content of the medium (Woolley and White, 1943). The inhibition index is given in Table 39. The efficiency of pyrithiamine as a thiamine antagonist is related to the specific vitamin requirements of the organisms tested. The inhibition index was low for those species which require intact thiamine, intermediate for those which require either or both moieties, and high for self-sufficient species. Table 39. The Efficiency of Pyrithiamine as an Inhibitor of Fungus and Bacterial Growth (Woolley and White, Jour. Exptl. Med. 78, 1943.) Organism Inhibition index pyrithiamine thiamine Thiamine requirement Ceratastomella fimbriata C. penicillata Phytophthora cinnamomi 7 10 12 11 130 800 800 400,000 40,000 5,000,000 Intact thiamine Intact thiamine Intact thaimine Chalaropsis thielavioides Intact thaimine Fndomyces vernalis Pyrimidine Mucor ramannianus Thiazole Saccharomyces cerevisiae Neurospora crassa Lactohacillus arabinosus L. casei Both moieties None None None Pyrithiamine was found to inhibit sporulation of Ceratostomella fimbri- ata, Choanephora cucurhitarum, and Chaetomium convolutum (Lilly and Barnett, 1948). This inhibition was overcome by thiamine. Pyrithia- mine was reported to be a more efficient antagonist for diphosphothia- mine than for thiamine when Penicillium digitatum was used as a test organism (Sarett and Cheldelin, 1944). METABOLITE ANTAGONISTS 233 Pyrithiamine causes a thiamine deficiency disease in mice, which may be cured or prevented by the administration of sufficient thiamine (Wool- ley and White, 1943). Neopyrithiamine is reported to be four times as active as pyrithiamine for the rat (Wilson and Harris, 1949). Biotin antagonists. Many biotin vitamers are known which are highly specific. The efficiency of an antibiotin in some instances may depend upon whether biotin or one of its vitamers is the competing metabolite. The formulas of two of the compounds are given below. Compare with the formulas of biotin and desthiobiotin given in Chap. 9. CO CO / \ / \ HN NH HN NH CHs— CH Cn(CH.2)5— SO3H H2C CH(CH2)5— COOH Sulfonic-acid analogue of desthiobiotin Imidazolidonecaproic acid Desthiobiotin and imidazolidonecaproic acid differ only by a methyl group. Desthiobiotin was found to act as a biotin vitamer for Saccha, romyces cerevisiae and other yeasts (Dittmer et al., 1944; Lilly andLeonian- 1944), while imidazolidonecaproic acid is an antibiotin for S. cerevisiae (Dittmer and Du Vigneaud, 1944). Both compounds are antibiotins for Lactobacillus casei. The sulfonic-acid analogue of desthiobiotin was shown by Duschinsky and Rubin (1948) to be more active against desthio- biotin and oxybiotin than against biotin for S. cerevisiae. The replace- ment of a carboxyl group by a sulfonic-acid group appears to be a rather general method of changing a metabolite into an antagonist. Further examples of this will be cited in connection with pantothenic and amino- acid antagonists. Egg white contains a specific protein which combines with biotin and thus renders this vitamin inactive. This inactivity is due to the molecu- lar size of the avidin-biotin complex, which prevents its absorption by organisms. Raw egg white may be used to produce experimental biotin deficiency in animals. Avidin is no longer active after heating, and bound biotin is released by this treatment. This specific protein has been used to separate biotin vitamers into two groups, for avidin com- bines only with those compounds w^hich have an intact urea ring structure. The papers of Eakin et al. (1941) and Burk and Winzler (1943) may be consulted for further details. Pantothenic acid antagonists. Yeasts are the only fungi which have been reported to be deficient for pantothenic acid, and in most instances /3-alanine replaces the intact vitamin molecule. One of the commonly studied pantothenic acid antagonists is the compound called pantoyl- taurine. The formulas for pantothenic acid and pantoyltaurine are given belo'rt 234 PHYSIOLOGY OF THE FUNGI HO— CH2— C(CH3)2- HO— CH2— C(CH3)2- CHOH— CO— NH- Pantothenic acid -CHOH— CO— NH- Pantoyltaurine CH2— CHo— COOH -CH2— CH2— SO3H Pantoyltaurine is the sulfonic-acid analogue of pantothenic acid. Snell (1941) studied the competitive inhibition of yeast growth by pan- toyltaurine and found that this compound was effective when pantothenic acid was the metabolite supplied in the medium but that pantoyltaurine did not compete with /3-alanine. The data in Table 40 illustrate this difference. Table 40. The Effect of Pantoyltaurine on the Growth of Saccharomyces cerevisiae in the Presence of Pantothenic Acid and (3-Alanine Inoculum used, 0.02 mg., time of incubation, 16 hr. (Snell, Jour. Biol. Chem. 141, 1941.) Calcium pantothenate, yug/lO ml. Sodium salt of pantoyl- taurine, Mg/10 ml. Moist cells, mg/10 ml. /3- Alanine, Mg/10 ml. Sodium salt of pantoyl- taurine, Mg/10 ml. Moist cells, mg./lO ml. 0.0 0 0.3 0.0 0 0.03 0.5 0 6.6 0.3 0 2.8 0.5 1,000 2.9 0.5 0 5.5 0.5 5,000 0.4 0.3 1,000 3.0 0.5 10,000 0.3 0.3 10,000 3.0 30.0 10,000 6.6 0.5 5,000 5.5 0.5 10,000 5.7 The synthesis of pantothenic acid via /3-alanine by Escherichia colt is inhibited by cysteic acid (sulfonic-acid analogue of aspartic acid). This inhibition is reversed by /3-alanine or pantothenic acid (Ravel and Shive, 1946). For further information concerning other pantothenic acid and other antagonists, the review of Roblin (1946) should be consulted. Pyridoxine antagonists. Some of the pyridoxine analogues studied by Robbins and Ma (1942) inhibited the growth of Ceratostomella ulmi. This inhibition was reversed by additional pyridoxine. The substitution of an ethyl group for the methyl group of pyridoxine produced an antag- onist for C. ulmi, but ethyl pyridoxine was as active for excised tomato roots as pyridoxine itself. The above authors suggest that ethyl pyri- doxine might be a chemotherapeutic agent for the Dutch elm disease. The formulas of ethyl pyridoxine and desoxypyridoxine are given below : HO- C2H2- /^-CHoOH -CH2OH HO- /%— CH2OH \n^ CH3— Ethyl pyridoxine Desoxypyridoxine METABOLITE ANTAGONISTS 235 Martin et at. (1948) found desoxypyridoxine to be slightly more effec- tive against pyridoxal than pyridoxine, when Saccharomyces cerevisiae was used. Vitamin K antagonists. There are at least two naturally occurring compounds which have vitamin K activity. Certain synthetic analogues are used in medicine to replace the natural vitamins. All these com- pounds are substituted 1,4-naphthoquinones. The structural formula for vitamin K2 is given below: O O -CHs CH3 CH3 I I -CHo— (CH=C— CH2— CH2)6— CH=C— CH3 Vitamin K2 Horsfall (1945) has reported 2-methyl-l,4-naphthoquinone to be a weak fungicide, although this compound replaces natural vitamin K in medi- cine. On the other hand, 2,3-dichloro-l,4-naphthoquinone (Phygon) is a potent fungicide (Ter Horst and Felix, 1943). O 0 — CHa O 2-Methy 1- 1 , 4-naphtho quinone —CI —CI o 2 , 3-Dichloro- 1 , 4-naphtho quinone Phygon may act as a fungicide by virtue of combination of the quinone with free amine or sulfhydryl groups. This mechanism probably inac- tivates certain enzymes noncompetitively. On the other hand, Phygon is structurally related to vitamin K, and a competitive type of inhibition should also be possible. Woolley (1945) investigated the inhibitory effect of 2,3-dichloro-l,4-naphthoquinone and 2-methyl-l,4-naphthoquinone on the growth of Saccharomyces cerevisiae and Endomyces vernalis. The first compound was more toxic than the second. In less than toxic concen- trations, the second compound partially overcame the toxicity of the first. The amount of 2,3-dichloro-l,4-naphthoquinone required to inhibit yeast (half maximum growth) was 1.7 jug per liter, while 230 ng of 2-methyl-l,4-naphthoquinone were required to produce the same amount of inhibition. Some of Woolley 's data are presented in Table 41. Many potent antimalarial drugs are 1,4-naphthoquinone derivatives (Fieser et al., 1948). It has been assumed in our discussion of the effects of antagonists on 236 PHYSIOLOGY OF THE FUNGI organisms that antimetabolites are active by virtue of interfering with various enzymatic processes. It is also interesting to note that com- petitive inhibition has been demonstrated with isolated enzyme systems, Schopfer and Grob (1949) found the action of urease to be inhibited by 2-chloro-l,4-naphthoquinone. Most of the activity was restored by the addition of 2-methyl-l,4-naphthoquinone (vitamin K3). Table 41. The Reversal of Inhibition Caused by 2,3-Dichloro-1,4-naphtho- QuiNONE by 2-Methyl-1,4-naphthoquinone Test fungus, Saccharomyces cerevisiae. Concentration of 2,3-dichloro-l,4-naphtho- quinone, 0.005 jug/ml. (Woolley, Proc. Soc. Exptl. Biol. Med. 60, 1945. Published by permission of the Society for Experimental Biology and Medicine.) 2-methyl-l,4- Turbidity naphthoquinone, ^g/ml. (100 = no growth) 0.0 93 0.04 60 0.02 68 0.01 77 0.005 85 Other vitamin antagonists. The sulfonic-acid analogue of nicotinic acid inhibits the growth of certain bacteria (Mcllwain, 1940). Appar- ently this analogue has not been tested in nicotinic acid-deficient fungi. Woolley (194Ga) has reported maize to contain a "pellagragenic" agent which may tentatively be considered as a naturally occurring anti- nicotinic-acid factor. Among the recently developed insecticides, 7-hexachlorocyclohexane is of considerable value. Kirkwood and Phillips (1946) have shown that the growth of Saccharomyces cerevisiae is inhibited by this compound, and that the inhibition is overcome by meso-inositol. The other isomers of hexachlorocyclohexane were not very effective inhibitors of yeast growth ; neither are they of much value as insecticides. These observations point to competitive inhibition as a possible mechanism of insecticidal action of this compound. AMINO-ACID ANTAGONISTS Organisms must either synthesize or obtain from exogenous sources the different amino acids they require for the synthesis of protein. Anti- metabolites which antagonize the synthesis or utilization of essential amino acids would have a profound effect upon growth or other functions of organisms. The role of amino acids is not confined to the synthesis of proteins but extends to the synthesis of other essential metabolites. An amino-acid antagonist may act in two ways, (1) by inhibiting protein synthesis and (2) by inhibiting the synthesis of essential metabolites which are derived from amino acids, either directly or indirectly. If an amino a^id functions in more than one way, the action of an amino- METABOLITE ANTAGONISTS 237 acid antagonist may be overcome, at least in part, by the action of second- ary metabolites as well as the primary metabolite. The toxic effect of 3-acetylpyridine on rats is reversed by either nicotinic acid amide or tryptophane (Woolley, 1945a). Analogues. Mitchell and Niemann (1947) found that the halogenated derivatives of phenylalanine and tyrosine competitively inhibit growth of the wild strain of Neurospora crassa (Table 42). The most effective of these inhibitors was 3-fiuoro-DL-phenylalanine. The structural form- ulas for this analogue and the natural metabolite are shown below: ^^-CHo— CHNH2— COOH /\ CHo— CHNH2— COOH V Phenylalanine 3-Fluorophenylalanine Table 42. Inhibition of Growth of Neurospora crassa by Some Halogenated Alpha-amino Acids Basal medium contained 30 mg. of DL-phenylalanine or 20 mg. L-tyrosine per liter depending upon the antagonist tested. (Mitchell and Niemann, Jour. Am. Chem. Sac. 69, 1947. Published by permission of the American Chemical Society.) Compound Mg./ml. for 50% inhibition Moles inhibitor Moles amino acid 3-Fluoro-DL-phenylalanine 3-Fluoro-DL-tyrosine 3-Fluoro-L-tyrosine 3-Fluoro-D-tyrosine 0.04 0.23 0.15 0.41 1.2 10.5 6.8 18.5 The other halogen derivitives (chloro, bromo, and iodo) were less effective inhibitors. 3-Fluorophenylalanine was shown to be an effective inhibitor for various other fungi and bacteria. The effect of j8-2-thienylalanine on the growth of a strain Saccharomyces cerevisiae and certain bacteria has been studied by Ferger and Du Vigneaud (1948). The formula for this thiophene analogue of phenyl- alanine is given below: HC C— CHo— CHNH2— COOH HC CH i3-2-Thienylalanine Only the l isomer is active in competing with phenylalanine. The replace- ment of divalent sulfur ( — S — ) by a vinylene group ( — CH=CH — ), or vice versa, often leads to the production of an antimetabolite. As another example, the effect of replacing sulfur in cysteine by radicals 238 PHYSIOLOGY OF THE FUNGI containing the vinylene group may be cited. Dittmer et al. (1948) found methallylglycine, allylglycine, and crotylglycine to inhibit the growth of Saccharomyces cerevisiae and Escherichia coli. The effects of these three antimetabohtes on the growth of yeast are shown in Fig. 49. 50 100 150 200 250 300 Micrograms of unsoturoted amino acids per 7.5 ml. Fig. 49. The inhibition of growth of Saccharomyces cerevisiae, strain 139, by DL-allyl- glycine, DL-methallylglycine, and DL-crotylglycine. (Courtesy of Dittmer, Goering, Goodman, and Cristol, Jour. Am. Chem. Soc. 70: 2501, 1948. Published by permis- sion of the American Chemical Society.) Natural amino acids. Antagonism among the amino acids is not limited to competitive inhibition between naturally occurring amino acids and their analogues. Robbins and McVeigh (1946) found hydroxy- proline to inhibit the growth of several dermatophytes: Tricho-phyton mentagrophytes, T. gypseum (granular form), T. purpureum, Epidermo- phyton fiocculosum, and Microsporum canis. This inhibition was over- come by proline. The relationship of these two naturally occurring amino acids is shown below: H2C- H.C -CH2 CH— COOH HOHC- H2C -CHo in- COOH N H Proline N H Hyd ro xy proline Low concentrations of hydroxyproline stimulated growth of Tricho- phyton purpureum, while higher concentrations inhibited growth. Addi- tion of hydroxyproline to a glucose-asparagine medium increased the growth of Polyporus squamosiis. Hydroxyproline was without effect on the growth of 19 other species of fungi. Whether amino-acid antago- nisms may limit the nitrogen utilization of natural mixtures of these METABOLITE ANTAGONISTS 239 compounds is unknown, but the possibility of inhibition should be kept in mind when only a few amino acids are used in a medium. The effect of any single compound upon a fungus may be modified by the other constituents of the medium. Harteiius (1946a) found glutamic and aspartic acids, glutamine, and asparagine to inhibit the growth of a strain of yeast when suboptimumal amounts of /^-alanine were used in the medium. These amino acids did not inhibit growth when pantothenic acid was used. In fact, these com- pounds stimulated growth under these conditions. The inhibitory effect in the presence of /3-alanine was overcome by increasing the concentration of this provitamin. To obtain maximum growth in the presence of 50 mg. of glutamic acid per flask (55 ml.), twenty times as much j9-alanine was required as when glutamic acid was omitted from the medium. Harteiius attributed this effect to the combination of /3-alanine and glutamic acid to form an inactive dipeptide. Among the naturally occurring amino acids, L-canavanine is found free in jack beans. Canavanine is an analogue of arginine; the formulas are shown below. HjN— C(=NH)— NH— CH2— CH2— CH,— CH(NH2)— COOH Arginine H2N— C(=NH)— NH— O— CH2— CH2— CHCNH,)— COOH Canavanine Horowitz and Srb (1948) studied the effect of canavanine on three wild-type strains of Neurospora and found one strain to be inhibited completely by concentrations greater than 1.25 mg. per liter; another strain was only partially inhibited, while the third strain was tolerant. Genetic analysis indicated that tolerance and susceptibility segregated by alternative forms of a single gene. L-Arginine was effective in over- coming canavanine toxicity, while L-lysine was less effective. Three molecules of arginine overcame one molecule of canavanine in the strain of Neurospora most sensitive to this inhibitor. A similar competitive inhibition between canavanine and arginine in various bacteria has also been observed (Volcani and Snell, 1948). Other metabolite antagonists. Woolley (1944a) found benzimidazole to inhibit the growth of Saccharomyces cerevisiae and Endomyces vernalis. This inhibition was overcome by adenine and guanine. The structural relationship between benzimidazole and adenine is shown below: N=C— NH2 HC C— NH \ CH — C— N Benzimidazole Adenine 240 PHYSIOLOGY OF THE FUNGI DEVELOPMENT OF FASTNESS An organism which has become tolerant, or resistant, to an inhibitor (analogue, drug, antibiotic, etc.) after exposure is said to be fast, or more specifically pyrithiamine-fast, sulfanilamide-fast, or penicillin-fast, as the case may be. Fastness is a very common phenomenon, although it appears to have been but little studied in fungi. It is an important factor which limits the use of many antibiotics and the sulfonamides in medicine. This phase of fungus physiology deserves more attention than it has received. It is conceivable that the prolonged use of a single fungicide to control a fungus pathogen could lead to the development, or selection, of a strain which would be relatively tolerant to the effect of the fungicide. Such findings do not appear to have been reported from field studies, but this possibility should be kept in mind. Fungi do become fast to various antagonists. WooUey (19446), by repeatedly subculturing Endomyces vernalis in a medium containing pyrithiamine, developed a strain which withstood twenty-five times the concentration of pyrithiamine which served to reduce the growth of the parent strain to half the maximum. In this instance, fastness was correlated with the ability of the pyrithiamine-fast strain to cleave the inhibitor molecule into its cyclic moieties. Thus, the development of pyrithiamine fastness may be ascribed to the formation of an adaptive enzyme which destroyed the antagonist. Escherichia coU, which is not inhibited by pyrithiamine, also hydrolyzed this compound. These results indicate that adaptive enzymes may play a role in the develop- ment of fastness. In addition to resistance or fastness which develops in organisms cul- tured in the presence of an inhibitor, it has been found recently that various bacteria not only develop resistance but may develop strains which are actually dependent upon the presence of the "inhibitor" before they can grow. Yegian et al. (1949) have found that culturing Myco- bacterium tuberculosis in the presence of streptomycin gave rise to strains which were fast to this antibiotic and also produced strains which cannot grow unless streptomycin is present in the medium. SUMMARY The normal utilization of a metabolite may be prevented or inhibited in three ways: (1) destruction or removal in an unavailable combination of a metabolite; the enzymatic hydrolysis of thiamine and the combina- tion of biotin with avidin are representative examples of this mode of inactivation ; (2) the noncompetitive inhibition of various enzymes by such compounds as iodoacetate, cyanide, and azide; (3) competitive inhibition due to metabolite antagonists. This type of inhibition is METABOLITE ANTAGONISTS 241 overcome by increasing the concentration of the normal metabohte. Antagonists are known which inhibit the functioning of vitamins, amino acids, and other metabohtes. It is postulated that a metabolite and its antagonists compete for the active surface of specific enzymes. The ratio of inhibitor to metabolite required to reduce growth to one-half its normal value is called the inhibi- tion index. Effective inhibitors have small inhibition indexes. The same compound may act as an antagonist for some fungi and as a metabo- lite for others; e.g., desthiobiotin. The medium used for investigating inhibition is important, for the presence of a normal metabolite, or a secondary metabolite derived from it, may prevent inhibition, A given compound may be considered as an antagonist, but it is only an antagonist for certain species, and then only under certain conditions. Organisms may acquire a tolerance or resistance to inhibitory agents and become fast. In extreme instances they become dependent upon the inhibitor, which then acts as a kind of growth factor. The competitive nature of many inhibitions is firmly established. In most instances there is a close structural relation between a metabolite and its antagonists. The theories which have been advanced to explain these phenomena have been useful in correlating the results of research and for increasing our insight into metabolic processes, REFERENCES BuRK, D., and R. J. Winzler: Heat-labile, avidin-uncombinable, species-specific and other vitamers of biotin, Science 97 : 57-60, 1943. DiTTMER, K., and V. du Vigneaud: Antibiotins, Science 100: 129-131, 1944. DiTTMER, K., H. L. GoERiNG, I. GooDMAN, and S. J. Cristol: The inhibition of microbiological growth by allylglycine, methallylglycine and crotylglycine, Jour. Am. Chem. Soc. 70: 2499-2501, 1948. DiTTMER, K., D. B. Melville, and V. du Vigneaud: The possible synthesis of biotin from desthiobiotin by yeast and the antibiotin effect of desthiobiotin for L. casei, Science 99 : 203-204, 1944. DuscHiNSKY, R., and S. H. Rubin: The synthesis and biological activity of 4-methyl- 5-(e-sulfoamyl)-2-imidazolidone, a sulfonic acid analog of desthiobiotin, Jour. Am. Chem. Soc. 70 : 2546-2547, 1948. Eakin, R. E., E. E. Snell, and R. J. Williams: The concentration and isolation of avidin, the injury-producing protein in raw egg white, Jour. Biol. Chem. 140: 535-543, 1941. Ferger, M. F., and V. du Vigneaud: The microbial growth inhibition produced by optical isomers of /3,2-thienyl-alanine, Jour. Biol. Chem. 174 : 241-246, 1948. FiESER, L. F., et al.: Naphthoquinone antimalarials. I. General survey, Jour. Am. Chem. Soc. 70: 3151-3155, 1948. FiLDES, P. : A rational approach to research in chemotherapy, Lancet 1 : 955-957, 1940. FouRNEAU, E., J. Tr^ifouel, J. Tri^fouel. F. Nitti, and B Bovet: Action du para- aminophenylsulfamide sur les moisisseures, Compt. rend, s jc. biol. 122 : 652-654, 1936. 242 PHYSIOLOGY OF THE FUNGI Hartelius, v.: Antivitamin effect of sulfanilamide on Aspergillus niqer, Conipl. rend. trav. lab. Carlsberg, Sir. physiol. 24: 178-184, 1946. Hartelius, V.: Glutamic acid, aspartic acid, asparagine and glutamine as anti- growth substances for /3-alanine, Conipt. rend. trav. lab. Carlsberg. SSr. physiol. 24: 185-222, 1946a. Hartelius, V., and K. Roholt: Effect of marfanil on Streptobacterium planatarum, Saccharomyces cerevisiae and Aspergillus niger, Compt. rend. trav. lab. Carlsberg, Sir. physiol. 24: 163-171, 1946. Horowitz, N. H., and A. M. Srb: Growth inhibition of Neurospora by canavanine and its reversal. Jour. Biol. Chern. 174: 371-378, 1948. HoRSFALL, J. G.: Fungicides and Their Action, Chronica Botanica Co., Waltham, 1945. KiRKWOOD, S., and P. H. Phillips: The anti-inositol effect of 7-hexachlorocyclo- hexane, Jour. Biol. Chem. 163: 251-254, 1946. Krampitz, L. O., and D. W. Woolley: The manner of inactivation of thiamine by fish tissue, Jour. Biol. Chem. 152: 9-17, 1944. Lampen, J. O., and M. J. Jones: The antagonism of sulfonamide inhibition of certain lactobacilli and entrococci by pteroylglutamic acid and related compounds, Jour. Biol. Chem. 166 : 435-448, 1946. Lilly, V. G., and H. L. Barnett: The effect of pyrithamin upon sporulation of three thiamin-deficient fungi. Am. Jour. Botany 35: 801, 1948. Lilly, V. G., and H. L. Leonian: The anti-biotin effect of desthiobiotin, Science 99 : 205-206, 1944. Martin, G. J., S. Avakian, and J. Moss: Studies of pyridoxine displacement, Jour. Biol. Chem. 174 : 495-500, 1948. *McElroy, W. D.: The mechanism of inhibition of cellular activity by narcotics, Quart. Rev. Biol. 22: 25-58, 1947. McIlwain, H.: Pyridine-3-sulphonic acid and its amide as inhibitors of bacterial growth, Brit. Jour. Exptl. Path. 21 : 136-147, 1940. McIlwain, H.: Interrelations in microorganisms between growth and metabolism of vitamin-like substances. Advances in Enzymol. 7 : 409-460, 1947. Mitchell, H. K., and C. Niemann: The competitive inhibition of the metabolism of a-amino acids by their halogenated analogs, Jour. Am. Chem. Soc. 69: 1232, 1947. MuDD, S.: Can chemotherapy be extended to include intra-cellular disease agents?. Jour. Bad. 49: 527-537, 1945. Ravel, J. M., and W. Shive: Biochemical transformations as determined by compe- titive analogue-metabolite growth inhibitions. IV. Prevention of pantothenic acid synthesis by cysteic acid. Jour. Biol. Chem. 166: 407-415, 1946. Robbins, W. J.: The pyridine analog of thiamin and the growth of fungi, Proc. Natl. Acad. Sci. U.S. 27: 419-422, 1941. Robbins, W. J., and R. Ma: Specificity of pyridoxine for Ceratostomella ulmi, Bull. Torrey Botan. Club 69: 342-352, 1942. Robbins, W. J., and I. McVeigh: Effect of hydroxyproline on Trichophyton menta- grophytes and other fungi. Am. Jour. Botany 33 : 638-647, 1946. RoBLiN, R. O., Jr.: Metabolite antagonists, Chem. Revs. 38: 255-377, 1946. ■*RoBLiN, R. O., Jr.: Metabolite antagonists. The relation between structure and biological activity, Chem. Eng. Neivs 27 : 3624-3629, 1949. RuBBo, S. D., and J. M. Gillespie: Para-aminobenzoic acid as a bacterial growth factor. Nature 146: 838-839, 1940. Rydon, H. N. : Anthranilic acid as an intermediate in the biosynthesis of tryptophane by Bact. typhosum, Brit. Jour. Exptl. Path. 29 : 48-57, 1948. METABOLITE ANTAGONISTS 243 Sarett, H. p., and V. H. Cheldelin: Inhibition of utilization of thiamine and diphosphothiamine for the growth of microorganisms, Jour. Biol. Chem. 156 : 91-100, 1944. ScHOPFER, W. H., and E. C. Grob: Ueber den Einfluss von 1,4-Naphtochinon- derivaten mit Vitamin-K- oder Antivitamin-K-Character auf die Urease, Helv. Chim. Acta 3^ : 829-838, 1949. Sealock, R. R., a. H. Ln'ERMORE, and C. A. Evans: Thiamine inactivation by the fresh-fish or Chastek-paralysis factor. Jour. Am. Chem. Soc. 65: 935-940, 1943. Sevag, M. G., R. a. Richardson, and J. Henry: Studies on the action of sulfo- namides on the respiration and growth of bacteria. IIB. Effect of serum on the inhibition of respiration and growth of Pneumococcus, Type I, and Staphylo- coccus aureus by sulfonainides and p-aminobenzoic acid. Jour. Bad. 49 : 139-147, 1945. ■^Shive, W., and J. Macow: Biochemical transformations as determined by competi- tive analogue-metabolite growth inhibitions. I. Some transformations involv- ing aspartic acid, Jour. Biol. Chem. 162 : 451-462, 1946. Singer, T. P. : Enzyme inhibitors and the active groups of proteins. Brewers Digest 20: 85-88 104-106, 1945. Snell, E. E.: Growth inhibition by N-(Q;,7-dihydroxy-/3,^-dimethylbutyryl)taurLne and its reversal by pantothenic acid, Jour. Biol. Chem. 141: 121-128, 1941. Stoddard, E. M.: The x disease of peach and its chemotherapy. Conn. Agr. Expt. Sta. Bull. 506, 1947. Ter Horst, W. p., and E. L. Felix: 2,3-Dichloro-l,4-naphthoquinone, a potent fungicide, Ind. Eng. Chem. 35: 1255-1259, 1943. Volcani, B. E., and E. E. Snell: The effect of canavanine, arginine and related compounds on the growth of bacteria, Jour. Biol. Chem. 174 : 893-902, 1948. Welch, A. D.: Interference with biological processes through the use of analogs of essential metabolites, Physiol. Revs. 25: 687-715, 1945. Weswig, p. H., a. M. Freed, and J. R. Haag: Antithiamine activity of plant mate- rials. Jour. Biol. Chem. 165 : 737-738, 1946. Wilson, A. N., and S. A. Harris: Synthesis and properties of neopyrithiamine salts, Jour. Amer. Chem. Soc. 71: 2231-2333, 1949. Wolf, F. T. : The action of sulfonamides and antibiotic agents on the pathogenic fungi in Biology of Pathogenic Fungi (edited by W. J. Nickerson), Chronica Botanica Co., Waltham, 1947. Woods, D. D. : The relation of p-aminobenzoic acid to the mechanism of the action of sulphanilamide, Brit. Jour. Exptl. Path. 21 : 74-90, 1940. WooLLEY, D. W.: Some new aspects of the relationship of chemical structure to biological activity, Science 100 : 579-583, 1944. WooLLEY, D. W.: Some biological effects produced by benzimidazole and their reversal by purines. Jour. Biol. Chem. 152 : 225-232, 1944a. WooLLEY, D. W. : Development of resistance to pyrithiamine in yeast and some observations on its nature, Proc. Soc. Exptl. Biol. Med. 55: 179-180, 1944b. *WooLLEY, D. W. : Observations on the antimicrobial action of 2,3-dichloro-l,4- naphthoquinone and its reversal by vitamins K, Proc. Soc. Exptl. Biol. Med. 60 : 225-228, 1945. WooLLEY, D. W.: Production of nicotinic acid deficiency with 3-acetylpyridine, the ketone analogue of nicotinic acid. Jour. Biol. Chem. 157 : 455-459, 1945a. WooLLEY, D. W. : Some aspects of biochemical antagonism in Currents in Bio- chemical Research (edited by D. E. Green), Interscience Publishers, Inc., New York, 1946. 244 PHYSIOLOGY OF THE FUNGI Wool LEY, D. W.: The occurrence of a "pcllagragenic" agent in corn, Jour. Biol. Chem. 163 : 773-774, 1946a. WooLLEY, D. W., and A. G. C. White: Selective reversible inhibition of microbial growth with pyrithiamine, Jour. Exptl. Med. 78 : 489-497, 1943. • Wright, L. D.: The significance of anti-vitamins in nutrition. Jour. Am. Dietet. Assoc. 23: 289-298, 1947. Yegian, D., V. BuDD, and R. J. Vanderlinde: Streptomycin-dependent bacilh: A simple method for isolation, Jour. Bad. 58 : 257-259, 1949. CHAPTER 12 THE ACTION OF FUNGICIDES The never-ending warfare which man must wage against parasitic fungi in order to protect his crops has been ably chronicled by Large (1940). The saprophytic species which decay wood and other cellulosic materials cause great economic loss, although these species perform a necessary and indispensable role in maintaining the carbon cycle in nature. It is to man's interest and profit that the deterioration of textiles and lumber be prevented or delayed and that his crops be protected from pathogenic fungi. This is done by the use of fungicides, which either kill or inhibit the action of fungi. By definition, an agent which kills fungi is a fungicide. A fungistatic agent merely causes inhibition. The same agent is commonly capable of producing both actions. A discussion of the terms fungicidal and fungi- static is given by McCallan and Wellman (1942). These authors point out that the fungistatic activity of an agent is broader than its fungicidal activity. Both physical and chemical agents may be fungicidal and fungistatic. Of the physical agents, heat and ultraviolet radiation are probably most commonly used, while many chemical compounds are ''toxic" to fungi. Whether an agent is fungicidal or fungistatic is primarily a matter of degree of intensity and duration of exposure. We may assume that these agents, whether chemical or physical, act directly upon certain specific enzymes or enzyme systems. If the action is less severe and may be reversed, the result is fungistasis, while if it is irreversible, the action is fungicidal. Since most of the agents employed by man are chemical compounds, much of the following discussion will be limited to the mechanism of action of these compounds. Chemical fungicides may be applied as eradicants or as protectants. A protectant is applied to the plant or other material before the inoculum arrives at the infection court and often functions only after the fungus spore germinates. An eradieant kills the fungus already present on or in the substrate material. The lethal action of a chemical depends upon both the concentration of the active compound or ion and the time of exposure. Species of fungi exhibit great variation in ability to resist the action of certain fungicides. Many fungi are killed by exposure to a few parts per million 245 246 PHYSIOLOGY OF THE FUNGI of cupric ion, while a few species have been reported to grow in a saturated solution of copper sulfate (Starkey and Waksman, 1943). There is no useful universal fungicide. The intelligent choice of a fungicide depends upon a number of factors, the major ones being the species of fungus to be controlled and the nature of the material to be protected. The solubility of the fungicide is of great importance. For most efficient preservation of wood or protection as a spray, a fungicide must have a low solubility in order that the protection may extend over a long period of time. For surface sterilization a highly soluble fungicide is used. When a fungicide is to be used on a living plant (or other organism), the relative sensitivity of the host and of the fungus to the fungicide must be considered. Host sensitivity limits the use of many potent fungicides. A useful fungicide must be more toxic to the fungus than to the host. For example, copper fungicides are quite toxic to cabbage, cucumber, and pea seed, while beet, eggplant, pepper, and spinach seed are relatively tolerant to copper. Although there is an enormous accumulation of literature on fungicides, their composition, application, limitations, and economic value (see Frear, 1948, and Horsfall, 1945), relatively little has been published on the mechanism of fungicidal action. This is a practical as well as an academic question, for the intelligent use of known fungicides and the search for new and better ones are based upon a knowledge of how they act. In the past the most important inorganic fungicides have contained compounds of copper, mercury, or sulfur. In the future, however, excel- lent fungicides may be made from other toxic elements. For example, cadmium is of potential interest, but the present supply is limited. In controlling fungi and other pests, there is always the danger that they will become tolerant, or fast, to a given toxicant. This means that the more susceptible individuals are killed and that a greater amount of a given fungicide is required to control the more tolerant population which is then built up. It is desirable from several viewpoints to have satis- factory reserve fungicides in the armory of the plant pathologist. COPPER The first copper salts to be used as fungicides were the sulfate and acetate (Prevost, 1807). These salts are soluble, and even in low con- centration they are too toxic for many uses. Since all the copper is available at once, these salts are toxic to plants, especially to young parts. These soluble salts have a further disadvantage when used as a spray, for a heavy dew or rain will easily wash them off. However, these salts, especially copper sulfate, were successfully used for treating seed grain to destroy surface contaminants. This treatment was devised by Prevost to control bunt. ACTION OF FUNGIDICES 247 The next advance in copper fungicides was not until 1885, whei Millardet published the formula for making the famous fungicide Bordeaux mixture. Millardet recommended that 8 kg. of copper sulfate pentahydrate (bluestone) be dissolved in 100 liters of water. This solu- tion was then mixed with 15 kg. of quicklime slaked in 30 liters of water. The chemistry of Bordeaux mixture is more complicated than was assumed at first. Instead of cupric hydroxide, a series of basic sulfates are formed, the composition being dependent upon the ratio of coppei sulfate and calcium hydroxide used (Frear, 1948). Bordeaux mixture is a copper compound or compounds of low solubility. According to Goldsworthy and Green (1936), Bordeaux mixture in equilibrium with water yields a solution containing about 4 p. p.m. of copper. However, McCallan and Wilcoxon (1936) found that well-washed 4-4-50 Bordeaux mixture was soluble only to the extent of furnishing 1 p. p.m. of copper. After this material was thoroughly dried, as in a spray film, the solubihty in terms of copper decreased to 0.2 to 0.3 p. p.m. McCallan and Wilcoxon have reported a comparison between the amounts of Bordeaux mixture and copper sulfate required to inhibit the germination of 90 per cent of the spores of a few species. These data are given in Table 43. Table 43. The Relative Efficiency of Bordeaux Mixture and Copper Sulfate IN Inhibiting Spore Germination (McCallan and Wilcoxon, Contribs. Boyce Thompsori Inst. 6, 1936.) Cu, mg. /liter, for LD 90 Species Bordeaux mixture Copper sulfate Uromyces caryophyllinus Sclerotinia fructicola 180 120 390 500 2,400 1.74 1 20 Botridis paeoniae 2 23 Glomerella cingulata Alternaria solani 1.40 6.72 If Bordeaux mixture or other copper spray or dust of low solubility furnishes less than 1 p. p.m. of copper to the solutions with which it is in equilibrium, it is obvious that the concentration of copper is too low for any great amount of toxicity. We must also take into account the rate ^ of solubility of the ''insoluble" copper compounds, for if the rate of solution is slow, the maximum concentration may not be attained in time to prevent infection. The only hypothesis which would account for the lethal action of copper compounds of such low solubility would be that of cumulative action. A germinating spore in a saturated solution of the copper compound in equilibrium with the solid copper compound would remove copper from the solution. This process would cause more 248 PHYSIOLOGY OF THE FUNGI of the copper compound to dissolve until the spore was no longer able to take more copper from the solution. This theory is attractive because of its simplicity, but there seems to be no very good evidence for it (McCallan, 1929). In practice, Bordeaux mixture and other "insoluble" copper sprays act as if they were more soluble than is indicated by chemical tests. However, in practice the fungicide is exposed to the action of the atmos- phere, the host plant, and the fungus spores. This is a more complicated situation than that found in the chemical determination of solubility. Barker and Gimingham (1911) found that intact leaves increased the soluble copper from Bordeaux mixture to some extent but were of the opinion that the host plant had only a slight influence on the solubility of such sprays. However, if the leaves were injured, they were quite effective in bringing copper into solution. The possibility that the spores exert a solvent action on "insoluble" copper compounds has long been considered by plant pathologists. The spores of at least some species do exert a solvent action on Bordeaux mixture. McCallan and Wilcoxon (1936) showed that the amount of copper brought into solution by the soluble materials washed from or excreted by 100 million spores of some species varied as follows: Uromyces caryophyllimis, 1.01 mg.; Sclerotinia fructicola, 0.76 mg.; Neurospora sitophila, 0.12 mg.; Botrytis paeoniae, 0.10 mg.; Glomerella cingulata, 0.046 mg. ; Aspergillus niger, 0.023 mg.; and Alternaria solani, 0.013 mg. Enough spores of Neurospora sitophila were collected so that the nature of the soluble materials from the spores could be identified chemically. Malic acid was isolated and identified. The presence of amino acids also was detected. Both malic acid (or malates) and various amino acids dissolve "insoluble" copper compounds under nevitral or alkaline conditions with the formation of soluble complex copper compounds. McCallan and Wilcoxon showed that sodium cuprimalate and a copper-glycine compound were about as toxic as copper sulfate. On the other hand, Goldsworthy and Green (1936) were of the opinion that spore secretions played a minor role in increasing the solu- bility of Bordeaux mixture, but the evidence of McCallan and Wilcoxon seems quite conclusive. Basic copper carbonate (malachite, Cu(OH)2-CuC03) and cuprous oxide (CuiO) are used in treating seeds. Since the seed covered with these materials is planted in soil which contains a variety of protein degradation products, it is easy to understand how these substances are made sufficiently soluble to be fungicidal. Marten and Leach (1944) studied the effect of various nitrogenous compounds upon the solubility of cuprous oxide. Gelatin and peptone were less efficient in dissolving cuprous oxide than were glycine, aspartic acid, asparagine, or cystine. Ammonium hydroxide was also a solvent for cuprous oxide. With all ACTION OF FUNGICIDES 249 these "solvents" the solutions were blue in color, which indicates that the copper was oxidized to the cupric state. Marten and Leach investi- gated the toxicity of the copper-glycine compound to Pythnim debaryanum. It was noted that an excess of glycine protected the fungus from the action of the copper. Some 200 times as much copper was required to inhibit growth when glycine was present in the medium as when it was absent. Thus, it seems that whether a given amount of copper is toxic or not depends upon the nature and amount of certain constituents in the medium or substrate. One may ask, By what mechanism does the copper ion cause fungistasis, or how does the copper kill? The common explanation of the toxic action of the heavj'^ metals (copper, mercury, and silver) is based upon the property of these ions of precipitating or denaturing proteins. Enzymes are proteins, and it would be expected that the heavy metals would inactivate these catalysts. However, not all enzymes are equally inac- tivated by low concentrations of heavy-metal ions. The enzymes which require free sulfhydryl groups for activity appear to be especially suscepti- ble to inactivation by ions of heavy metals. It is probable that copper causes fungistasis by combining with the sulfhydryl groups of certain enzymes. At this stage, the action of copper is reversible. Goldsworthy and Green (1936) found that spores of Sderotinia fructicola which had been treated with insufficient copper to kill made normal growth when sown on copper-free medium. As long as an inhibition is reversible, the process is one of fungistasis. Death of the spore results when irreversible changes occur. There is reason to believe that the injurious effect of copper fungicides upon the host plant is due to the same mechanism that operates in fungus spores. Foster (1947) attributed the sensitivity to copper of certain seeds to their content of sulfhydryl enzymes. mercury' While a number of inorganic salts of mercury have been used as anti- septics, only two have had wide application as fungicides. Mercuric chloride (corrosive sublimate, bichloride of mercuiy, HgCl2) is a soluble, highly poisonous compound. It is commonly used for surface steriliza- tion in a concentration of 1/1,000. Mercuric chloride is occasionally used as a special-purpose fungicide. Mercurous chloride (calomel, HgCl or Hg2Cl2) is essentially insoluble in water, sufficiently so to be used in medicine. Calomel slowly decom- poses into mercury and mercuric chloride. This decomposition is accel- erated by sunlight, which may account for the successful use of calomel to control dollar spot, brown patch, and other turf diseases. The organic mercury compounds have won wide acceptance in the 250 PHYSIOLOGY OF THE FUNGI treating of seed to control the attack of fungi which cause damping-off and of certain seed-borne pathogens. The organic mercurials are free from many of the objections inherent in the inorganic compounds of mercury. In general, they combine less avidly with proteins, are more selective in their action, and are far less toxic to animal life. As used for seed protection, they are commonly diluted with an inert carrier. Most if not all such organic mercury compounds are sold under trade names, but the active components are required by law to be stated on the label. Among these organic mercury compounds are ethylmercury phosphate (Semesan Jr. and New Improved Ceresan), ethylmercury chloride (Ceresan) and hydroxymercurichlorophenol (Semesan). The organic mercury compounds used as sprays and for treating seeds are in general related to mercuric chloride in the following way: C2H6— Hg— CI CI— Hg~Cl Ethylmercury chloride Mercuric chloride The ethyl group has replaced a chlorine atom in mercuric chloride. The type formula for compounds like this may be written as R — Hg — X, where R may be any alkyl or aryl (or other) group and X represents any anion, I~, Cl~, 0H~, NOs", P04~. The anion greatly modifies the solu- bility of the compound in water. In general, this type of organic mer- cury compound is volatile, and this property may be assumed to aid in penetration. Other organic mercury fungicides are derivatives of alkjd and aryl mercuric hydroxides. These compounds can react with organic acids to form salts. The relation of these compounds to mercuric hydrox- ide is shown below: CeHs— Hg— OH HO— Hg— OH Phenylmercury hydroxide Mercuric hydroxide Compounds of this type are used to protect cellulose and leather products. Parker-Rhodes (1942) investigated the toxicity of the following mer- cury compounds to Macrosporium sarcinaeforme and Botrytis allii: mer- curic acetate, mercuric chloride, methylmercury nitrate, and tolylmer- curic nitrate. All these compounds were toxic to M. sarcinaeforme, and all except methylmercury nitrate were toxic to B. allii. Perhaps methyl- mercury nitrate is not soluble enough in fat for the spores of this fungus to absorb a toxic amount of the compound. Dillon- Weston and Booer (1935) found that vapor of ethylmercury iodide was toxic to TiUetia spores in the laboratory but afforded no control in the field. The soluble inorganic mercury salts are protein precipitants, and this property may explain in part their mode of action when used in high concentrations. These salts are frequently fungistatic or bacteriostatic, since the very firmness of the union between the mercuric ion and the cell membrane may form a barrier to further penetration. The first action ACTION OF FUNGICIDES 251 of mercuric ion is to cause stasis, which may be reversed by treating the cells with reagents which have a high affinity for mercury. McCalla (1940) demonstrated that cells of Escherichia coli which had been treated with mercuric chloride could be revived by hydrogen sulfide. If stasis due to mercury is not overcome within a certain time, irreversible changes occur and death of the cells results. Organic mercury compounds are not protein precipitants, and this is one of their advantages as disinfectants and fungicides. Fildes (1940) ascribed the action of mercury compounds to combination of mercuric ion with the sulfhydryl group of essential metabolites and enzymes. Others have shown that organic mercury compounds act similarly. According to this view, enzyme inhibition is the basis of the action of mercury compounds. Fildes found that the action of mercury was antagonized by compounds which contained free sulfhydryl groups (thioacetate, cysteine, glutathione). Neither cystine ( — S — S — ) nor methionine ( — S — ) was effective in overcoming mercury toxicity. Organic mercury compounds appear to act by the same mechanism as the mercuric ion. p-Chloromercuribenzoate was found to inhibit the action of various sulfhydryl enzymes which take part in carbohydrate metabolism, e.g. succinic acid oxidase, yeast carboxylase, malate oxidase, and ketoglutarate oxidase. This organic mercury compound also inhib- ited the action of c?-amino acid oxidase, transaminase, Z-glutamate oxidase, and other enzymes (Barron and Singer, 1945; Singer and Barron, 1945). In many instances the inhibitory action of p-chloromercuribenzoate on these enzymes could be reversed by glutathione, cysteine, or hydrogen sulfide. Cook et at. (1946) found phenylmercuric nitrate to depress the respira- tion of Saccharomyces cerevisiae. This depression in rate of respiration was overcome by various compounds having a free — SH group; e.g., cysteine and homocysteine, while cystine and methionine were without effect. The work of these investigators and of others makes it highly probable that mercury compounds are toxic because they inactivate certain essential enzyme systems. The enzyme inhibitions discussed above are examples of noncompetitive inhibition. These inhibitions are reversible, as in the case of competitive inhibition, but the reversing agents are nonspecific, or not limited to a single metabolite. SULFUR Of the nonmetallic elements, sulfur and certain of its compounds are widely used as protective and eradicant fungicides. The toxicity of the nonmetallic elements is dependent upon the state of oxidation. In many instances, compounds in the higher states of oxidation are the least toxic. For example, sulfur in the form of the free element (S) and of sulfide 252 PHYSIOLOGY OF THE FUNGI (S=) forms excellent fungicides, while sulfites (SOa^) are only slightly- toxic and sulfates (SO 4=) are nontoxic. According to Large (1940), elemental sulfur has been used to control powdery mildew for slightly over 100 years. The effectiveness of sulfur increases as the particle size diminishes. Finely divided sulfur adheres to plant surfaces much better than larger particles. In addition, the distance between particles tends to be decreased when fine particles are used, and the infection court is thereby better protected. The odds are increased that a fungus spore falling upon a treated leaf will be within the range of action of a particle of sulfur. An example of the effect of particle size of sulfur on toxicity is given in Table 44. The greater toxicity of Table 44. The Relation between the Particle Size and Toxicity of a Sulfur Dust to the Conidia of Sclerotinia americana (Wilcoxon and McCallan, Phytopathology 20, 1930.) Treatment Mean diameter of sulfur particles, tx Germination, % Control 285 142 60 33 97.6 Ground roU sulfur 62.8 Ground roll sulfur Ground roll sulfur Ground roll sulfur 47.2 29.1 20.7 the finely divided sulfur is due to the fact that sulfur enters the spore in the form of vapor. The amount of vapor formed from a given amount of sulfur in a given time depends upon the area of the exposed surface, as well as upon temperature. Therefore, the fineness of the sulfur particles governs the effective concentration of sulfur vapor and its effectiveness as a fungicide. McCallan (1946) estimated the yearly consumption of sulfur in the United States alone to be 142 million pounds. Of this amount 110 million pounds is used as sulfur dust, 5 million pounds as wettable sulfur, and 27 million pounds as lime-sulfur. Approximately 62 per cent of this is used primarily for the control of apple scab alone. Since elemental sulfur is insoluble, its action upon fungi cannot be attributed to the sulfur in this form. Two general theories have been proposed to explain the action of sulfur. One theory holds that the action is due to oxidized sulfur, such as SO2 or SO3 (which form sulfurous and sulfuric acid, respectively, with water) or pentathionic acid, H2S5O6. According to the second theory, the reduced form of sulfur, H2S, is the active toxic agent. Both these theories are supported by published experimental evidence. All these compounds are toxic to fungi under certain conditions, if in high enough concentrations. However, to ACTION OF FUNGICIDES 253 account satisfactorily for the toxic properties of sulfur, it must be demon- strated that the toxic agent is produced under the conditions which prevail in the field in quantities sufficient to account for the observed effects. Any evaluation of these hypotheses must take into account all the variables involved. The caution of Wilcoxon and McCallan (1930) is pertinent: In making comparisons of the toxicity of chemical substances to fungus spores, there are two requisites for obtaining accurate results which, though quite obvious, have not always received the consideration they deserve, (a) The substance whose toxicity is to be measured must be available in a pure state and of known concentration, and (6) the technique employed must be capable of distinguishing between the toxicity of the substances it is desired to compare. It is agreed that elemental sulfur is not the toxic agent and that sulfur is transformed into the toxic agent. There are three possible agencies for such transformations: the atmosphere, the plant on which the sulfur is dusted, and the fungus spores or mycelium. Sulfur acts at a distance, and since sulfur is volatile at room temperature, this property offers an explanation. Sulfur vapor is a gas, and in this state it should be more easily transformed into the toxicant. Sulfur is slowly oxidized by the oxygen of the atmosphere to form sulfur dioxide, but the rate at which this reaction occurs at ordinary tempera- tures makes it impossible for this reaction to account for all the toxic properties of sulfur, even though sulfur dioxide is toxic to fungus spores (McCallan and Weedon, 1940). Young (1922) set forth the hypothesis that pentathionic acid is the toxic agent formed from sulfur. It is agreed, even by those who do not support Young's hypothesis, that this acid is formed on the surface of sulfur dust. A considerable number of papers were published during the next decade which gave support to this view (Liming, 1932). Wil- coxon and McCallan (1930) investigated this theory thoroughly and concluded that pure pentathionic acid had no toxic properties for the spores of Sderotinia americana, Botrytis sp., Macrosporium sarcinaeforme, and Uromijces caryopJujUinus. If sufficient pentathionic acid was used to reduce the pH to about 4, spore germination was inhibited. Solutions of sulfuric acid having the same pH were equally toxic. Neutral salts of both acids were nontoxic. Roach and Glynne (1928) likewise found pentathionic and sulfuric acids to have the same toxicity when tested against the winter sporangia of Synchytrium endohioticum. Wilcoxon and McCallan (1930) performed a decisive experiment when they washed one lot of sulfur dust with alkali to remove pentathionic acid and com- pared this pentathionate-free dust with the original sample, which con- tained a trace of this acid. No difference in toxicity of the washed and control samples of this sulfur dust was found. 254 PHYSIOLOGY OF THE FUNGI There is now general agreement that hydrogen sulfide is the common toxic compound produced from sulfur. Not only is hydrogen sulfide toxic to fungus spores, but the mechanism for its production is also pres- ent. It is known that hydrogen sulfide is produced from sulfur both by the treated plant and by the fungus spores, McCallan and Wilcoxon (1931) made qualitative tests for the ability of the spores of 17 species of fungi to produce hydrogen sulfide from sulfur. All produced this substance, but in varying amounts and at varying rates. They showed that the spores need not be in direct contact with solid sulfur to produce hydrogen sulfide. Figure 50 illustrates the method used by these investigators to demonstrate this phenomenon. These authors investigated the toxicity of hydrogen sulfide to the spores of eight species of fungi. These experiments were performed in a flowing stream of air which con- tained known amounts of hydrogen sulfide, and the concentration in the water droplet in which the spores were suspended was calculated from Henry's law. These precautions are necessary because hydrogen sulfide is unstable. Neglect of this fact by LEAD ACETATE PAPER WHITE LEAD ACETATE PAPER BLACKENED COLLODION SAC SPORE SUSPENSION SULPHUR PASTE Fig. 50. The production of hydrogen sulfide by Sclerotinia spores separated from sulfur by a collodion membrane. Note that the production of hydrogen sulfide takes place on the spore side of the membrane and not on the sulfur side. (Courtesy of McCallan and WUcoxon, Contribs. Boyce Thompson Inst. 3: 26, 1931.) earlier investigators led to an underestimation of the toxicity of hydrogen sulfide. These results of McCallan and Wilcoxon are presented in Fig. 51. From these curves it is seen that spores of Venturia inaequalis, Uromyces caryophyllinus, and Puccinia antirrhini are inhibited by very low concentrations of hydrogen sulfide, while the spores of Botrytis sp. and Glomerella cingulata are scarcely affected by ten times as much hydrogen sulfide. By increasing the hydrogen sulfide concentration to 60 p. p.m., complete inhibition of germination of the spores of these two species was obtained. The spores of these eight fungi were shown to produce varying amounts of hydrogen sulfide per unit weight of spores. Whether hydrogen sulfide produced by spores would prove toxic would therefore depend upon the ability of the particular spores to produce hydrogen sulfide and the sensitivity of the spores to this substance. The correlation is shown in Table 45. The actions of sulfur and hydrogen sulfide are parallel, and it may be concluded that sulfur is toxic to the spores of certain species by virtue ACTION OF FUNGICIDES 255 of absorption of sulfur vapor and its reduction to hydrogen sulfide within the spore. Thus, the spores of susceptible species destroy themselves. It is not thought that the hydrogen sulfide evolved from leaves or other spores is absorbed in lethal quantities under natural conditions. 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Concentration -milligrams per liter Fig. 51. Toxicity of hydrogen sulfide to urediospores of Uromyces caryophyllinus and Puccinia antirrhini and to conidia of Venturia inaequalis, Sclerotinia americana, Macrosporiuni sarcinaeforme, Pestalotia stellata, Glonierella cingulata, and Botrytis sp. (Courtesy of McCallan and Wilcoxon, Contribs. Boyce Thompson Inst. 3: 31, 1931.) Liquid lime-sulfur is a common spray material and is prepared by boiling sulfur and calcium hydroxide together. The chief active ingre- dient is calcium polysulfide. After deposition on leaves the calcium polysulfide is quickly decomposed, yielding sulfur and calcium sulfide, Table 45. Comparison between the Toxicity and the Production of Hydrogen Sulfide, Expressed in Units Equal to the Amounts of Hydrogen Sulfide Required to Reduce Germination 50 Per Cent (McCallan and Wilcoxon, Contribs. Boyce Thompson Inst. 3, 1931.) Species Mg. H2S required to reduce ger- mination of 1,000,000 spores 50% Mg. H2S produced by 1,000,000 spores in 12 hr. Production of H2S expressed in units equal to the amount of H2S required to reduce germination 50% Venturia inaeaualis 0.001 0.002 0.006 0.013 0.043 0.049 0.532 0.665 0.002 0.019 0.13 0.039 0.013 0.001 0.027 0.002 2.0 Uromyces caryophyllinus Puccinia antirrhini 9.5 2.2 Sclerotinia americana Macrosporiuni sarcinaeforme Pestalotia stellata Glonierella cingulata 3.0 0.30 0.02 0.05 Botrytis sp 0.003 256 PHYSIOLOGY OF THE FUNGI which in turn may decompose by hydrolysis to yield hydrogen sulfide and calcium hydroxide. At the same time some of the calcium poly- sulfide is oxidized to calcium thiosulfate and sulfur. See Frear (1948) for a discussion of the chemistry involved. It is known that lime-sulfur exerts an eradicant action on some fungi, including Venturia inaequalis, when first applied. After a few days this spray exerts only a protective action like that of elemental sulfur, which probably depends on the elemental sulfur set free by the decom- position of various constituents comprising lime-sulfur. The eradicant action, then, depends upon either the calcium polysulfide or calcium sulfide. We may consider that sulfide ion (S=) is the toxic agent. The alkalinity of the spray may aid in penetration into the mycelium already present. Lime-sulfur solution may be treated with ferrous sulfate or aluminum sulfate in the spray tank to produce colloidal sulfur and hydrogen sulfide. Aluminum sulfate, AI2 (804)3, hydrolyzes to form aluminum hydroxide and sulfuric acid. A lime-sulfur spray so treated has only a protective action. It has lost its eradicant value. We may assume, therefore, that the decomposition of lime-sulfur in spray tanks when treated with acid (aluminum sulfate) and the decomposition on the leaf follow a somewhat similar pattern. This scheme of producing colloidal sulfur has the draw- back that the added iron, when ferrous sulfate is used, is toxic to vegeta- tion, and dangerous amounts of hydrogen sulfide are evolved. We may assume that hydrogen sulfide exerts its toxic action on fungus spores by inactivating certain enzymes. Hydrogen sulfide is known to inactivate many enzymes, including catalase, cytochrome oxidase, dopa oxidase, lactase, and others. Generally, hydrogen sulfide and cyanide inhibit the same enzymes. It is thought that these metalloenzymes are inhibited by sulfide or cyanide because these agents react with iron or copper to form highly insoluble or little-ionized compounds or complexes. ORGANIC FUNGICIDES The newer fungicides, with few exceptions, are either organic or organo- metallic compounds. The organic mercury compounds were considered with the inorganic compounds of mercury, since the mechanism of action appears to be the same in both types of compounds. Many of the organic fungicides exhibit greater specificity than the inorganic fungicides. The possibilities of modification in the structure of organic compounds are almost unlimited. The study of organic fungicides, therefore, offers the opportunity of correlating structure with type and intensity of fungi- cidal action. Aldehydes. The first organic fungicide to attain wide acceptance was formaldehyde. At one time this compound was used for the surface sterilization of grain and potato tubers, but at present formaldehyde is ACTION OF FUNGICIDES 257 little used. Formaldehyde reacts with free amino groups, and it is probable that its fungicidal action depends upon this property. Some other aldehydes also have fungicidal properties (Uppal, 1926). Quinones. While there are two series of quinones (ortho, or 1,2, and para, or 1,4), we shall consider only the 1,4-quinones as fungicides. The simplest quinone is p-benzoquinone. Quinones are cyclic compounds which possess a characteristic pair of double bonds. Such a configuration of double bonds is called quinoid and is possessed by many dyes, some of which are fungicides. If a considerable series of toxic compounds possess a common functional group or groups, it may be assumed that these groups are involved in fungicidal activity. According to Horsfall (1945), 1,4-benzoquinone has a slight toxicity to fungi. The four hydro- gens in 1,4-benzoquinone can be replaced by chlorine to form chloranil (Spergon), which greatly increases the fungicidal properties. The struc- tural formulas for these compounds are given below: O O — H -H CI— Cl- — CI -CI o o 1,4-Benzoquinone Chloranil (Spergon) Spergon has been used as a seed protectant. Substituted naphthoquinones are more important fungicides than, the benzoquinones. Among these, 2,3-dichloro-l,4-naphthoquinone (Phygon) is reported to be five to eight times as effective as Spergon (Ter Horst and Felix, 1943). Some of the naphthoquinones synthesized by plants are fungicides. Juglone, 5-hydroxy-l,4-naphthoquinone, is found in walnut hulls and is secreted by walnut roots. The isomeric 2-hydroxy- 1,4-naphthoquinone (lawsone) is found in henna leaves. Juglone is reported to be as toxic to fungus spores as Bordeaux mixture. Juglone controls black spot of roses as well as sulfur does (Gries, 1943, 1943a). It is also toxic to many plants. Little et al. (1948) isolated 2-methoxy- 1,4-naphthoquinone from Impatiens balsamina. This compound was an active fungicide which exhibited no phytotoxicity toward tomato and bean plants. The formulas of two naphthoquinone fungicides are given below: O O -OCH3 o 2-Methoxy-l,4-naphthoquinone OH O Juglone 258 PHYSIOLOGY OF THE FUNGI The fungicidal action of substituted quinones may be due in part to their property of reacting with free amino groups of proteins (Theis, 1945). Substituted naphthoquinones as antagonists of vitamin K were discussed in Chap. 11. Most of the available evidence indicates that the principal mechanism of quinone toxicity lies in its noncompetitive inhibition of sulfhydiyl enzymes. It has been suggested that the mechanism of inhibition is dependent upon the structure of the substituted naphthoquinones. Colwell and McCall (1946) found the fungistatic and fungicidal concentrations of 2-methyl-l,4-naphthoquinones to be the same when Aspergillus niger and an unidentified fungus were used as test organisms. Addition of sodium thioglycolate or cysteine antagonized the toxic action of this naphthoquinone. These authors postulate that only naphthoquinones unsubstituted in position 3 react wdth sulfhydryl groups, for 2-methyl-3- methoxy-l,4-naphthoquinone was not antagonized by thioglycolate or cysteine. The reaction between certain naphthoquinones and sulfhydiyl- containing compounds can be demonstrated in vitro. The amounts of various substituted 1,4-naphthoquinones required to cause a 50 per cent inhibition of isolated yeast carboxylase and similar reduction in the germination of Monilinia fructicola spores w^ere roughly parallel (Foote et al., 1949). Carboxylase is a sulfhydryl enzyme. It is probable that other sulfhydryl enzymes are also inhibited by naphtho- quinones. For further information on the mechanism of quinone inhibi- tion, see Geiger (1946). Dyes. Various dyes are fungistatic compounds. Malachite green and crystal violet are used to control various fungus infections of the skin. Both these dyes have a benzoquinoid structure, as is shown below: ^ ^ -N(CH3)2 (CH3)2N=' / \/ ^ CI Malachite green Leonian (1930) made a study of the toxicity of malachite green to many species and strains of Phytophthora and found only three species {P. hydrophila, P. melongenae, and P. sp.) able to grow in the presence of 1 p. p.m. of malachite green. Other species were more sensitive to this dye. P. colocasiae and P. richardiae failed to grow in nutrient solutions containing 1 part of malachite green in 16 million parts of medium. Leonian (1932) investigated the growth-inhibiting properties of malachite ACTION OF FUNGICIDES 259 (CH3)2N =C N(CH3)2 N(CH3)2 Crystal violet CI green and crystal violet upon 26 species and isolates of Trichophyton. Malachite green proved greatly superior to crystal violet. Over half the isolates tested failed to grow in the presence of 1 part of malachite green to 50,000 parts of medium, and many failed to grow in the presence of 1 p.p.m. of this dye. Crystal violet allowed some growth in all isolates tested at a concentration of 1 part in 50,000 parts of medium. Placing the inoculum in direct contact with the medium containing the dye was more lethal than placing the agar inoculum plug with the mycelium upon the surface of the test medium. Some other dyes such as methylene blue are also toxic to fungi. Both malachite green and methylene blue inhibit carboxylase (Horsfall, 1945). Dithiocarbamates and related compounds. Barratt and Horsfall (1947) have reported extensive investigations on the homologues and analogues of disodium ethylenebisdithiocarbamate (Nabam). In gen- eral, these compounds are formed when primary and secondary amines react with carbon disulfide. The formula for Nabam is given below: S H H S Na— S— C— N— CH2— CH2— N— C— S— Na Disodium ethylenebisdithiocarbamate (Nabam) The zinc (Ziram) and ferric (Ferbam) salts of dimethyldithiocarbamate are effective fungicides for the control of certain fungus pathogens. The formula for dimethyldithiocarbamate is given below: S II (CH3)2— N— C— SH Dimethyldithiocarbamate The oxidation product of dimethyldithiocarbamate is tetramethylthluram disulfide (Thiram), which has some value as a seed protectant. The dithiocarbamate fungicides, such as Nabam, yield hydrogen sulfide on hydrolysis. This reaction takes place spontaneously in the presence of moisture. The mechanism of hydrogen sulfide toxicity has already been discussed. The second mechanism which has been proposed involves the formation of insoluble mercaptides of certain essential metals. 2G0 PHYSIOLOGY OF THE FUNGI In addition, Nabam on decomposition yields an unidentified toxic gaseous compound, which is neither hydrogen sulfide nor sulfur dioxide (Rich and Horsfall, 1950). Specific organic reagents for metals. The essential nature of certain micro elements for fungus growth and the role of these elements in enzymes were discussed in Chaps. 4 and 5. The chemistry of these specific organic reagents is treated by Yoe and Sarver (1941). These reagents form insoluble or slightly ionized compounds with metals. Zentmeyer (1944) tested various organic analytical reagents and found 8-hydroxyquinoline (Oxine) and ammonium nitrosophenylhydroxylamine (Cupferron) to be fungistatic. 8-Hydroxyciuinoline inhibited the growth of Fusarium oxysporum var. lycopersici, Ceratostomella ulmi, and a species of Penicillium. The effectiveness of 8-hydroxyquinoline in forming chelate salts increases as the pH values increase. Below pH 3.5 complex formation does not take place with zinc, copper, iron, and manganese. Zinc ion reacts with 8-hydroxyquinoline as shown below: + H+ I I r OH O— Zn 8-Hydroxyquinoline Zinc complex of 8-hydroxyquinoline The fungistatic effect of 8-hydroxyquinoline on Fusarium oxysporum var. lycopersici and Ceratostomella ulmi was overcome by increasing the zinc content of the medium. In the presence of 8-hydroxyquinoline there was competition between this compound and one or more enzyme systems for the zinc present in the medium. Whether or not an organic compound such as 8-hydroxyquinoline will act as a fungistatic agent depends upon the concentration of the reagent, the amount of fungus mycelium, and the concentration of the metallic ion for which the two sj^stems compete. One would expect that such fungicides, in common with all others, would be more effective when the mass of the fungus is small. Other organic fungicides. Many other types of organic compounds are fungicides, and an intensive search for new ones is in progress. Brief mention of some of these developments is made below. Geiger (1948) reports various unsaturated ketones to be active against Aspergillus niger, Trichoderma koningii, Cryptococcus neoformans, and Trichophyton mentagrophytes. The mode of action resembles that of the naphtho- quinones in that sulfhydryl enzymes, including succinic acid dehydro- genase, triose phosphate dehydrogenase, and urease, are inhibited. The fungistatic activity of ethylenic and acetylenic compounds has been ACTION OF FUNGICIDES 261 tested on Fusarium graminearum, Penicillium digitatum, and Botrytis aim (McGowan et al., 1948). The fungicidal action of substituted pyrazoles was tested on spores of Alter naria oleracea and Sclerotinia americana in the laboratory, and for the control of apple scab, cedar-apple rust, and late blight of potato and tomato. Some of these compounds show promise, although the mechanism of action is not known (McNew and Sandholm, (1949). For a survey of the newer fungicides see Well- man (1948). EVALUATING FUNGICIDES The preliminary tests of fungicidal activity are made in the laboratory in order to eliminate inactive compounds or to compare the activities of different compounds under identical conditions. Evaluation in the greenhouse and field is the final test of a new fungicide. This discus- sion will be limited to a general consideration of laboratory testing of fungicides. Fungus spores rather than mycelium are used in most laboratory tests because it is the function of a protectant fungicide to kill or inhibit spore germination. Three basic types of procedures may be used in laboratory tests (McCallan, 1947): (1) Spores are suspended in solutions or suspen- sions of the fungicide under test, and the inhibition of germination is noted as a function of time of exposure and concentration of the fungicide. This is a modification of the Rideal- Walker method of evaluating anti- septics. (2) The compound to be tested is incorporated in a suitable solid or liquid medium, which is then inoculated with spores of the test fungi. The amount of inhibition of germination or growth is determined. (3) Glass slides are covered uniformly with the fungicide, and after dry- ing, the spores are sown on the treated slides. The inoculated plates are then placed in constant-humidity chambers and the percentage of germination determined after 20 to 24 hr. ; or the effectiveness of a fungi- cide may be studied as a function of time of exposure. The second and third methods appear to be the most useful. Fleury (1948) studied the fungistatic action of thiourea on Aspergillus niger by adding this substance to a liquid basal medium. Thiourea was a much more potent inhibitor w^hen nitrate nitrogen was used than when ammonium or organic nitrogen was present in the medium. Agar medium has been used by Leben and Keitt (1949) to assay the amount of toxicant on leaf surfaces. A suspension of spores of Glomerella cingulata was prepared in warm (38 to 40°C.) agar medium. Five milliliters of this seeded medium was added to Petri dishes which contained 15 ml. of solidified agar medium. After the seeded agar had solidified, leaf disks of uniform size were cut from sprayed leaves and placed on the agar. The amount of toxicant present on the leaf surface was determined by measur- 262 PHYSIOLOGY OF THE FUNGI ing the diameter of the zone of inhibition. Disks of blotting paper to which fungicides have been added may be used to determine their potency. Thornberry (1950) has suggested the use of filter-paper disks for the evaluation of fungicides and bactericides. Filter-paper disks appear to be more suitable than blotting paper. In this method seven filter- paper disks are uniformly spaced on a Petri dish, and 0.09 ml. of the toxicant in aqueous solution is added per disk. The zone of inhibition is a measure of the effectiveness of the fungicide. The glass-slide method appears to simulate more closely the conditions under which the spores of plant pathogenic fungi germinate in nature. The Committee on the Standardization of Fungicidal Tests of the Ameri- can Phytopathological Society has considered this method important enough to publish a detailed and documented summary (1943), to which the student is referred for further information and references. This committee recommended the use of spores of the following species for this test: Alternaria solani, Glomerella cingulata, Macrosporium sarcinae- forme, Sclerotinia fructicola, Penicillium expansum, and Rhizopus nigri- cans. For accurate work, at least two of these test fungi should be used. The effectiveness of a fungicide is determined by calculating the percent- age of inhibition of spore germination. The methods of evaluating data obtained in fungicide tests are discussed by Horsfall (1945). SUMMARY A fungicide is an agent capable of killing some fungi. Fungicides may be either water-soluble or nearly insoluble. The action of fungicides of the first class is immediate; that of the second class is delayed. Eradi- cant fungicides are of the first class, w^hile protective fungicides are of the second. Fungistasis is the complete or partial inhibition of one or more life processes of a fungus. This inhibition is reversible. The same chemical compound may cause fungistasis or may be a fungicide, depend- ing upon the concentration and time of exposure. The same substance may be a fungicide for one species, cause fungistasis of a second, and be without effect upon a third. Fungistasis precedes fungicidal action. Before a fungicide can act upon a fungus, the toxicant must get into the fungus cells, or at least reach the protoplasmic membrane. While other factors undoubtedly enter into the mechanism of fungicidal action, the principal point of attack appears to be enzyme systems. The heavy- metal fungicides appear to act by inhibiting various sulfhydryl enzymes. Fungus spores transform sulfur into hydrogen sulfide, which inhibits the metalloenzymes. Organic fungicides, so far as is known, are also enzyme inhibitors. In the past, fungicides containing copper, mercury, and sulfur have been the most useful. Recently, organic fungicides have become impor- ACTION OF FUNGICIDES 263 tant and promise to be used even more extensively in the future. Organic fungicides are generally more specific than inorganic fungicides. Satis- factory fungicides for the control of certain diseases are still undiscovered. REFERENCES Barker, B. T. B., and C. T. Gimingham: The fungicidal action of bordeaux mix- tures, Jour. Agr. Set. 4: 76-94, 1911. Barratt, R. W., and J. G. Horsfall: Fungicidal action of metallic alkyl bisdi- thiocarbamates, Conn. Agr. Expt. Sta. Bull. 508, 1947. Barron, E. S. G., and T. P. Singer: Studies on biological oxidations. XIX. SuLf- hydryl enzymes in carbohydrate metabolism, Jour. Biol. Chem. 157 : 221-240, 1945. CoLWELL, C. A., and M. McCall: The mechanism of bacterial and fungus growth inhibition by 2-methyl-l,4-naphthoquinone, Jo^ir. Bad. 51: 659-670, 1946. *CoMMiTTEE ON THE STANDARDIZATION OF FUNGICIDAL Tests: The slide-germination method of evaluating protectant fungicides, Phytopathology 33 : 627-632, 1943. Cook, E. S., G. Perisutti, and T. M. Walsh: The action of phenylmercuric nitrate. II. Sulfhydryl antagonism of respiratory depression caused by phenylmercuric nitrate, Jour. Biol. Chem. 162 : 51-54, 1946. Dillon-Weston, W. A. R., and J. R. Booer: Seed disinfection. 1. An outline of an investigation on disinfectant dusts containing mercury. Jour. Agr. Sci. 26 : 628-649, 1935. FiLDES, P.: The mechanism of the anti-bacterial action of mercury, Brit. Jour. Exptl. Path. 21 : 67-73, 1940. Fleury, C.: Action de la thio-uree sur V Aspergillus niger. Role particulier joue par la source d'azote nitrique, Bull. soc. botan. Suisse 58 : 462-476, 1948. FooTE, M. W., J. E. Little, and T. J. Sproston: On naphthoquinones as inhibitors of spore germination, Jo2ir. Biol. Chem. 181: 481-487, 1949. Foster, A. A.: Acceleration and retardation of germination of some vegetable seeds resulting from treatment with copper fungicides, Phytopathology 37 : 390-398, 1947. Frear, D. E. H.: Chemistry of Insecticides, Fungicides and Herbicides; 2d ed., D. Van Nostrand Company, Inc., New York, 1943. Geiger, W. B. : The mechanism of the antibacterial action of quinones and hydro- quinones. Arch. Biochem. 11 : 23-32, 1946. Geiger, W. B.: Antibacterial unsaturated ketones and their mode of action. Arch. Biochem. 16: 423-435, 1948. GoLDSwoRTHY, M. C, and E. L. Green: Availability of the copper of Bordeaux mixture residues and its adsorption by the conidia of Sclerotinia fructicola, Jour. Agr. Research 52: 517-533, 1936. Gries, G. A.: Juglone (5-hydroxy-l,4-naphthoquinone) — a promising fungicide, Phytopathology 33: 1112, 1943. Gries, G. A.: Juglone — the active agent in walnut toxicity. Northern Nut Growers Assoc. Ann. Kept. 34: 52-55, 1943a. *HoRSFALL, J. G.: Fungicides and Their Action, Chronica Botanica Co., Waltham, 1945. Large, E. C: The Advance of the Fungi, Henry Holt and Company, Inc., New York, 1940. Leben, C, and G. W. Keitt: Laboratory and greenhouse studies of antimycin preparations as protectant fungicides. Phytopathology 39 : 529-540, 1949. 264 PHYSIOLOGY OF THE FUNGI Leonian, L. H. : Differential growth of Phytophthoras under the action of malachite green, Am. Jour. Botany 17: C71-677, 1930. Leonian, L. H.: Effects of position of inoculum on growth of some Trichophytons in the presence of dyes, Arch. Dermatol, and Syphilol. 25: lOlG-1020, 1932. Liming, O. N.: The relation of pentathionic acid and its component constituents to the toxicity of sulphur fungicides, Phytopathology 22: 143-165, 1932. *LiTTLE, J. E., T. J. Sproston, and M. W. Foote: Isolation and antifungal action of naturally occurring 2-methoxy-l,4^naphthoquinone, Jour. Biol. Chem. 174: 335-342, 1948. McCalla, T. M.: Cation adsorption by bacteria, Jour. Bad. 40: 23-32, 1940. *McCallan, S. E. a.: Studies on fungicides. III. The solvent action of spore excretions and other agencies on protective copper fungicides, Cornell Univ. Agr. Expt. Sta. Mem. 128, 1929. McCallan, S. E. a. : Outstanding diseases of agricultural crops and uses of fungi- cides in the United States, Contribs. Boyce Thompson Inst. 14: 105-115, 1946. McCallan, S. E. A.: Bioassay of agricultural fungicides, Agr. Chemicals 2(9): 31-34, 67; 2(10): 45, 1947. McCallan, S. E. A., and F. R. Weedon: Toxicity of ammonia, chlorine, hydrogen cyanide, hydrogen sulphide, and sulphur dioxide gases. II. Fungi and bacteria, Contribs. Boyce Thompson Inst. 11 : 331-342, 1940. McCallan, S. E. A., and R. H. Wellman: Fungicidal versus fungistatic, Contribs. Boyce Thompson Inst. 12: 451-463, 1942. ■*McCallan, S. E. a., and F. Wilcoxon: The fungicidal action of sulphur. II. The production of hydrogen sulphide by sulphured leaves and spores and its toxicity to spores, Contribs. Boyce Thompson Inst. 3: 13-38, 1931. McCallan, S. E. A., and F. Wilcoxon: The action of fvmgous spores on Bordeaux mixture, Contribs. Boyce Thompson Inst. 6: 151-165, 1936. McGowan, J. C, P. W. Brian, and H. G. Hemming: The fungistatic activity of ethylenic and acetylenic compounds. I. The effect of the affinity of the sub- stituents for electrons upon the biological activity of ethylenic compounds, Ann. Applied Biol. 35: 25-36, 1948. McNew, G. L., and N. K. Sandholm: The fungicidal activity of substituted pyra- zoles and related compounds. Phytopathology 39 : 721-751, 1949. Marten, E. A., and J. G. Leach: Some factors influencing the solubility of cuprous oxide in relation to its toxicity as a fungicide, Phytopathology 34 : 459-470, 1944. Parker-Rhodes, A. F. : Studies on the mechanism of fungicidal action. IV. Mer- cury, Ann. Applied Biol. 29: 404-411, 1942. Provost, B. : Memoire sur la cause immediate de la carie ou charbon des bl^s, et de plusieurs autres maladies des plantes, et sur preservatifs de la carie, 1807. Trans, by G. W. Keitt, Phytopathological Classics No. 6, American Phyto- pathological Society, Menasha, 1939. Rich, S., and J. G. Horsfall: Gaseous toxicants from organic sulfur compounds, Am. Jour. Botany 37: 643-650, 1950. Roach, W. A., and M. D. Glynne: The toxicity of certain sulphur compounds to Synchytrium endobioticum, the fungus causing wart disease of potatoes, Ann. Applied Biol. 15: 168-190, 1928. Singer, T. P., and E. S. G. Barron: Studies on biological oxidations. XX. Sulf- hydryl enzymes in fat and protein metabolism. Jour. Biol. Chem. 157: 241-253, 1945. Starkey, R. L., and S. A. Waksman: Fungi tolerant to extreme acidity and high concentrations of copper sulfate, Jour. Bad. 45: 509-519, 1943. ACTION OF FUNGICIDES 265 Ter Horst, W. p., and E. L. Felix: 2,3-Dichloro-l,4-naphthoquinone, a potent fungicide, Ind. Eng. Chem. 35: 1255-1259, 1943. Theis, E. H.: The collagen-quinone reaction. 1. Fixation and thermolability as a function of pH values, Jour. Biol. Chem. 157: 23-33, 1945. Thornberry, H. H. : a paper-disk plate method for the quantitative evaluation of fungicides and bactericides. Phytopathology 40: 419-429, 1950. Uppal, B. N.: Toxicity of organic compounds to the spores of Phytophthora colocasiae Rac, Jour. Agr. Research 32 : 1069-1097, 1926. Wellman, R. H.: Synthetic chemicals for agriculture. II. Fungicides, nematocides, rodenticides and weed killers, Chem. hids. 63 : 223-229, 1948. WiLCOXON, F., and S. A. E. McCallan: The fungicidal action of sulphur. I. The alleged role of pentathionic acid, Phytopathology 20: 391-417, 1930. YoE, J. H., and L. A. Sarver: Organic Analytical Reagents, John Wiley & Sons Inc., New York, 1941. Young, H. C: The toxic property of sulphur, Ann. Missouri Botan. Garden 9: 403-435, 1922. *Zentmeyer, G. a. : Inhibition of metal catalysis as a fungistatic mechanism, Science 100 : 294-295, 1944. CHAPTER 13 METABOLIC PRODUCTS The most important product of fungus metabolism is carbon dioxide, and the most important function of the fungi in the economy of nature is the destruction of plant and animal remains. The use of fungi for food antedates written history. The use of fvmgi for the preparation of bread and wine developed as a household art. From the time of Pasteur, the study of fermentation has led to an ever-increasing knowl- edge and understanding of the activities of microorganisms. The pro- duction of antibiotics and vitamins, alcohol and organic acids, and the potential utilization of waste agricultural products are current fields of research and industrial activity. For extensive treatment of these subjects the reader is referred to Prescott and Dunn (1949) and Foster (1949). DECOMPOSITION OF ORGANIC MATERIALS Brefeld (1908) called fungi "Organismen der Verwesung" and con- sidered them to be indispensable agents in maintaining the essential- element balance of nature. Saprophytic fungi and bacteria prevent the accumulation of plant and animal debris and return the elements that compose these materials to the storehouse of nature, where they are reused by new generations of plants and animals. In this role, sapro- phytic fungi are designated as "vegetable vultures" by Rolfe and Rolfe (1926), for they act as scavengers in the plant world. Green plants assimilate carbon in the form of carbon dioxide. Waks- man (1938) has assembled the data with regard to the amount of carbon in the biosphere. It is estimated that the atmosphere contains 600 billion tons of carbon in the form of carbon dioxide, and plants are esti- mated to remove 16 billion tons yearly. Thus, the carbon content of the atmosphere is sufficient for about 40 years, if no carbon dioxide were returned to the air. The complete destruction of plant and animal remains by fungi and bacteria requires a long time, although some plant constituents, such as soluble sugars and other carbohydrates, are quickly utilized. Presum- ably the fungi are the most important organisms in this process. Other plant constituents, such as the waxes and lignin, are attacked more slowly. The more resistant constituents are slowly modified to form 266 METABOLIC PRODUCTS 267 humus. Some of the carbon and other essential elements is converted into bacterial and fungus protoplasm, which after death is subject to decay. In the end, humus is converted into carbon dioxide, water, and other simple compounds, which are used again. The importance of humus as a soil constituent is ably discussed by Waksman (1938). In addition to the carbon cycle, the fungi also play an important part in the cycles involving the release and utilization of the other essential elements. FUNGI AS FOOD Many curious details about the early use of fungi as food have been collected from classical and other sources by Buller (1914) and by Rolfe and Rolfe (192G). The mushrooms were no doubt among the first fungi used as food by man. Yeast became part of his diet when the arts of brewing and baking were discovered. The widespread use of fermented beverages, under certain dietetic circumstances, has an important bearing on nutrition and health. J. S. Wallerstein (1939) has discussed primitive brewing practices and the geographical distribution of the art. The beer of the Middle Ages Avas turbid, owing to its content of suspended yeast cells (Thaysen, 1943). The nutritive value of any food depends upon its composition and digestibility and the assimilability of its hydrolytic products. The early writers, in the absence of precise information, were of the opinion that fungi had little value as food. The nutritive value of fungi, of yeast in particular, will be discussed from the standpoint of protein content and value, vitamins, fats, and minerals. Assuming good digestibility, the value of fungus protein is determined by its amino-acid composition. Rose (1938), in a long series of careful experiments, has determined which amino acids are essential for man and animals. Some nine or ten amino acids were found to be essential (Table 20). If the protein part of a diet is deficient in a single essential amino acid, nitrogen is lost from the body, or inefficient utilization of protein results. More of a poor protein must be consumed in order to increase the intake of essential amino acids to satisfactory levels. The amino- acid composition of yeast and some other proteins is given in Table 46. Yeast protein compares favorably w4th casein or meat with respect to essential amino acids. Less complete data are available for the amino-acid composition of fleshy fungi. According to Lintzel (1941), the proteins of Psalliota campestris, Cantherella cibarius. Boletus edulis, and Morchella esculenta are about equal to animal protein. From 100 to 200 gr. (dry weight) of these mushrooms was required to maintain the nitrogen balance in a man weighing 70 kg. Fitzpatrick et al. (1946) found the tryptophane content of P. campestris to be 5 mg. per 100 g. 268 PHYSIOLOGY OF THE FUNGI Table 46. Approximate Amino-acid Composition (in Per Cent) of Some Plant AND Animal Proteins Calculated to 16 Per Cent Nitrogen (Block and Boiling, Arch. Biochem. 7, 1945. Published by permission of Academic Press, Inc.) Amino acid Arginine Histidine Lysine Tyrosine Tryptophane . Phenylalanine Cystine Methionine . . . Threonine. . . . Leucine Isoleucine .... Valine Yeasts * Meat Casein Corn gluten Max. Min. 5.3 3.1 7.7 4.1 3.1 3.1 2.3 2.9 2.5 1.6 8.1 6.7 7.2 7.5 0.8 3.7 3.4 3.4 6.4 6.7 1.5 1.2 1.3 1.2 0.7 4.6 2.9 4.9 5.2 6.4 1.1 0.9 1.3 0.4 1.1 2.8 2.6 3.3 3.5 4.0 6.0 5.1 5.4 3.9 4.1 8.5 6.1 7.7 12.1 24.0 6.2 5.5 5.2 6.5 5.0 5.9 4.6 5.7 7.0 5.0 Polished rice 7.2 1.5 3.2 5.6 1.3 6.7 1.4 3.4 4.1 9.0 5.3 6.3 * Eight strains analyzed. The value of fungus protein in nutrition can be assessed only in relation to the amino-acid composition of the remainder of the diet. If the dietary proteins are low in certain essential amino acids, the supplemen- tary value of yeast (or other) protein may be great. The cereal grains, which furnish the bulk of protein for the population of the world, are generally low in one or more essential amino acids. Usually cereal protein is low in lysine or tryptophane or both. Sure (1946, 1947) studied the effect on the growth of rats of adding 1, 3, and 5 per cent of dried yeast to diets which contained cereals as the sole source of protein. The most marked effect of yeast occurred on a maize diet. At the end of a 10-week experimental period the rats receiving only cereal weighed 27.3 g., while the rats which received an additional 1 per cent yeast weighed 50.5 g. Rats which received the cereal plus 3 and 5 per cent yeast weighed 91.8 and 109.9 g., respectively. The effect of yeast was not so great when wheat or rice supplied the protein in the diet. In general, the most promising use of yeast protein in human nutrition is as a supplement rather than as a sole source of protein. Yeasts are efficient in absorbing and concentrating the vitamins present in the media in which they grow (Gorcica and Levine, 1942). The relative value of yeast as a source of vitamins depends upon the vitamin content of the other constituents of the diet. The prevalence of vitamin deficiency diseases (beriberi, pellagra, and others) is evidence that the vitamin content of many diets is inadequate. METABOLIC PRODUCTS 269 A dramatic demonstration of the value of yeast as a source of vitamins is reported by Bray (1928), onetime medical officer, Nauru, Central Pacific. The mandating government prohibited the brewing of toddy (palm wine) and allowed the sale of refined sugar. The results of these dietary changes were appalling. Soon, 40 per cent of the infants born in 1 year perished of infantile beriberi (thiamine deficiency) before reach- ing the age of 6 months. The restoration of toddy and enforced con- sumption of the dregs, i.e., the yeast, reduced the incidence of beriberi to one death in 16 months. Truly, Bray was right in calling toddy the elixir of life of the Nauruans. Piatt and Webb (1945) have noted that a simple maize diet which was inadequate with respect to riboflavin and nicotinic acid was made adequate in these respects by converting a por- tion of the dietary maize into maize beer. The vitamin content of yeasts depends upon the species or strain and the conditions of cultivation. Some representative data are presented in Table 47. Table 47. Vitamin Content of Seven Food Yeasts Results in milligrams per 100 g. of dry yeast. (Von Loesecke, Jour. Am. Dietet. Assoc. 22, 1946. Published by permission of the American Dietetic Association.) Species Torula utilis Saccharomyces cerevisiae* S. cerevisiae S. cerevisiae f S. cerevisiae^ S. cerevisiae f S. cerevisiaeX Thiamine 1.7 17.0 20.5 17.5 17.5 16.0 3.0 Riboflavin 4.7 8.0 7.6 4.2 4.5 3.6 7.5 Nicotinic acid 19.0 25.0 29.0 48.0 37,0 32.0 38.0 Pantothenic acid 86.0 112.0 122.0 86.0 72.0 74.0 13.5 * Six per cent salt added. t Debittered brewer's yeast. t Primary yeast. The production of fats by fungi is discussed elsewhere in this chapter. The usual fatty acids, including palmitic and oleic acids, are found in fat synthesized by fungi. Apparently few studies have been made on the value of fungi as sources of fat and essential minerals in human nutrition. CULTIVATION OF FUNGI FOR FOOD The ants were perhaps the first to cultivate fungi as a source of food (see Leaoii, 1940, for discussion and references). Fungi have been used for centuries in the Orient as food for man. The Chinese grow Hirneola polytricha and the Japanese grow Armillaria shii-take on oak saplings. The mushroom cultivated almost exclusively in the Occident is Agaricus 270 PHYSIOLOGY OF THE FUNGI (Psalliota) campestris. The method of cultivating this species on com- posted horse manure was developed near Paris before 1700. For informa- tion on mushroom growing the reader is referred to Duggar (1915). While attempts to replace composted horse manure by other substrates have been made, none appears to be entirely satisfactory. Humfield (1948) has suggested that Psalliota campestris be grown in large fermentors and the mycelium rather than the fruit bodies be used for food. Aspara- gus butt juice, a waste agricultural product, is a suitable medium. The chemical composition of mycelium and that of the fruit bodies is similar and the flavor comparable. This approach perhaps offers a way to culti- vate other desirable species, including the morels and the truffles. Nord (1948) has suggested that the mycelium of Fusarium lini be used for food. The use of yeasts to convert low-grade carbohydrates, such as wood sugar and molasses, into food has interesting possibilities. It is necessary to fortify these carbohydrates with other nutrients for the cultivation of yeast. Phosphates, a source of potassium, and nitrogen, in the form of urea, ammonia, or ammonium salts, are added. The function of yeast is to convert inorganic nitrogen into protein. Animals are unable to assimilate ammonia or urea directly but require nitrogen in the form of protein or amino acids. Inorganic nitrogen may be converted into proteins by green plants or by certain microorganisms. The use of urea, a derivative of ammonia, as cattle fodder is an example of the synthesis of protein by the microflora of the rumen. The possibility of using wood waste for yeast propagation was investi- gated in Germany during the First World War. In 1944 it is reported that 9,000 tons of food yeast were produced in Germany. Fermentable carbohydrates are obtained from wood as a by-product of sulfite paper manufacture, or by direct hydrolysis. Before sulfite liquor or wood hydrolysate is used for yeast culture, it is treated with calcium carbonate to adjust the pH and precipitate impurities. After the addition of nutrients the solution is heavily inoculated with the desired strain of yeast. Aeration is necessary for high yields of yeast. The weight of yeast produced amounts to about half the weight of sugar utilized. Such yeast is approximately 50 per cent protein (Harris et al., 1948). The economics of fodder-yeast production from sulfite liquor have been studied by Schleef (1948). The use of by-product molasses for the production of food and fodder yeasts should offer fewer technical difficulties than the use of wood sugar. FAT PRODUCTION Serious efforts to utilize fungi for the synthesis of fats were made in Germany during the First World War and continued thereafter. The technical problems encountered proved difficult, but some success was achieved by 1942 (Hesse, 1949). The controHing factor in fat production METABOLIC PRODUCTS 271 appears to be the carbon-nitrogen ratio. As long as an adequate supply of nitrogen is present, little fat is synthesized. If the carbohydrate sup- ply is high when the nitrogen is exhausted, assimilable fat is synthesized. Linder (1922) termed these two phases 'protein generation and Jat genera- tion. Fat-laden cells of many fungi appear to be incapable of cell division. Fat formation takes place only in the presence of an abundant supply of oxygen. The relation between sugar concentration and amount of fat synthesized by Penicillium javanicum is illustrated in Fig. 52. C7> 2500 > ^ 1. 2000 Myce //um"'' N \ 40 > / \\ \ 1500 Per cent fat / \ \ 35 > / t \ innn / / i k 30 / ^ \ 3» E 200 300 400 Groms of glucose per liter 500 Fig. 52. The effect of the concentration of glucose on the amount of mycelium and amount of fat synthesized by PenicilUum javanimm cultured in 75 ml. of medium for 12 days. (Drawn from the data of Lockwood, Catholic Univ. of America Biol. Ser. 13, p. 8, 1933. Published by permission of the Catholic University of America.) Among the fungi investigated for fat synthesis are Endomyces vernalis, Oidium lactis, Tonda utilis, Rhodotorula ghdinis, and species of Aspergil- lus, Penicillium, Mucor, and Fusarium. From a practical standpoint, only fungi which are capable of synthesizing fat in submerged culture are of potential value. E. vernalis and 0. lactis do not produce fat effici- ently in submerged culture. The fat content of various filamentous fungi was determined by Preuss et al. (1934) and Ward et al. (1935). The use of E. vernalis for fat and protein synthesis has been reviewed by Raaf (1941). Starkey (1946) studied fat production by an unidentified soil yeast, which under favorable conditions contained from 50 to 63 per cent 272 PHYSIOLOGY OF THE FUNGI crude lipide. A list of species of Penicillium and Aspergillus which syn- thesize considerable fat is given in Table 48. Table 48. The Crude Fat Content op Dried Mycelium of Various Species op PeniciUiuin and Aspergillus as Determined by Extraction with Ether (Ward et al., Ind. Eng. Chem. 27, 1935. Published by permission of the American Chemical Society.) Species Crude Fat, % Penicillium flavo-cinereum 28.5 P. piscarum 26-28 P. oxalicum 24.4 P. roqueforti 22.9 P. javanicum 22 . 2 Aspergillus flavus 16.0 Various theories of the mechanism of fat synthesis have been published and are reviewed by Foster (1949) and Hesse (1949). Most of these consider acetaldehyde or acetate to be the product of intermediary metab- olism used in fat synthesis. This emphasizes the importance of pyruvic acid in fungus metabolism. Various investigators have shown that acetaldehyde may be converted into fat by yeasts. The glycerol required for fat synthesis is thought to arise from the reduction and hydrolysis of dihydroxyacetone phosphate or 3-phosphogly eerie aldehyde (scheme VI, Chap. 7). PRODUCTION OF VITAMINS Only a few species of fungi and bacteria produce vitamins in large enough amounts to be of interest in industry. Biological synthesis must compete with chemical synthesis on a cost basis. The recovery of vita- mins as a by-product of commercial processes or the use of waste materials as the basis of a cheap medium may make biological synthesis attractive. Riboflavin is produced so abundantly by Candida guilliermondi under certain cultural conditions that it crystallizes in the medium (Burkholder, 1943). Among the factors found to influence the amount of riboflavin synthesized, the sources of carbon and nitrogen and aeration are impor- tant. Various investigators have found the concentration of iron in the medium to have a profound influence on the amount of riboflavin syn- thesized by various organisms. Iron concentrations in excess of 10 ^g per liter decreased the amount of riboflavin synthesized by C. guillier- mondi and C. fiareri (Tanner et al., 1945; Tanner and Van Lanen, 1947). The optimum iron concentration for riboflavin synthesis by Clostridium, acetobutylicum is said to be 1 mg. per liter. Hickey (1945) has suggested the use of 2,2'-bipyridine to inactivate excessive concentrations of iron in industrial fermentations. By maintaining the iron concentration between 40 and 60 ng per liter, Levine et al. (1949) found the maximum yields of riboflavin produced by C. guilliermondi and C. fiareri to be 175 and 567 ng per ml., respectively. Pilot-plant yields were somewhat less. METABOLIC PRODUCTS 273 Eremothecium ashbyi was shown to produce as much as 157 mg. per liter of riboflavin when cultivated on glucose-peptone medium (Renaud andLachaux, 1945). Aeration was necessary. Foster (1947) has recom- mended a molasses medium for the commercial production of riboflavin by E. ashbyi. The closely related species, Ashbya gossypii, also synthe- sizes riboflavin in large amounts (Tanner et al., 1949). Peltier and Borchers (1947) determined the amount of riboflavin produced by 240 isolates of soil fungi when grown on wheat bran. Forty- five isolates produced 2 mg. or more of riboflavin per 100 g. of dry mold bran. An unidentified species of Aspergillus produced 5.8 mg. of ribo- flavin per 100 g. of substrate. Species of Fusarium and Aspergillus were outstanding producers of riboflavin. The commercial microbiological synthesis of riboflavin depends upon the use of either E. ashbyi or C. acetobutyliciim (Tanner et al., 1949). Vitamin B12 was isolated in crystalline form from liver and shown to contain cobalt (Rickes et al., 1948; Smith, 1948). It is the only vitamin so far discovered which contains a metal as an integral part of the mole- cule. Streptomyces griseus and other microorganisms synthesize this vitamin. Sheep and cattle pastured on cobalt-deficient soils (Florida, Australia, New Zealand) develop a deficiency disease. Ingested cobalt is more effective than injected cobalt in overcoming this condition. It may be assumed that cobalt is used in the synthesis of vitamin B12 by the action of the microorganisms of the rumen and intestine. Vitamin B12 appears to be the anti-pernicious-anemia factor (West, 1948). Whether it is the animal protein factor is undecided. The cow-manure factor may be vitamin B12 (Lillie et al., 1948). Until the structure of vitamin B12 is determined and methods of syn- thesis developed, certain natural products will remain the only source of this vitamin. The only organic moiety of vitamin B12 so far disclosed is l-a-D-ribofuranosido-5,6-dimethylbenzimidazole (Brink et al., 1950). Vitamin B12 is obtained as a by-product from various industrial processes, especially streptomycin production. It is evident that the medium must contain cobalt; within limits, the amount of vitamin B12 synthesized by Streptomyces griseus is a function of the cobalt content of the medium. Maximum synthesis was observed when the medium contained 1 to 2 mg. of cobalt per liter (Hendlin and Ruger, 1950). None of the other vitamins appears to be synthesized by fungi in amounts which would make the latter attractive sources for the isolation of pure vitamins. The value of these vitamins in fungi used for food was discussed previously. Yeast can be fortified with thiamine so that it may serve as a therapeutic agent. By adding synthetic thiamine to an aerated yeast culture, yeast was produced which contained 6 mg. of thiamine per g. (Van Lanen et al., 1942). 274 PHYSIOLOGY OF THE FUNGI ENZYME PRODUCTION The industrial production and use of enzymes from microorganisms in the Occident is fairly recent, although the use of fungi as amylolytic agents by the peoples of the Orient for the preparation of koji and other foods is an old art. For this purpose, mixed cultures of species of Asper- gillus and Rhizopus are grown upon the rice or soybean substrates, the enzymes being used without separation. The pioneering work of Taka- mine (1914) on the amylases of A. oryzae was especially important. The ability of fungi to produce amylase is widely distributed, but only a few species are used commercially for this purpose. The amount of amylase produced varies with the species or isolate and the cultural condi- tions. Le Mense et al. (1947) screened 359 isolates of Penicillium and Aspergillus and found 42 isolates to produce amylase in submerged culture. The activity of the species of Penicillium ranged from 0.1 to 0.6 enzyme unit per milliliter of culture medium. One isolate of A. niger (NRRL 337) was found to be especially adapted for the production of amylase in submerged culture. The production of amylase was highly dependent upon the composition of the medium. Corn meal was espe- cially valuable in increasing amylase production when added to basal media composed of corn steep liquor, dried tankage, soybean meal, or thin stillage. Amylase production was stimulated by the addition of 10 to 20 mg. of sodium chloride per liter of culture medium. Addition of a mixture of chlorinated phenols (Dowcide G) inhibited sporulation and increased amylase production (Erb et al., 1948). Others have found different isolates of the same species to produce varying amounts of amylase. Hao et al. (1943) studied the production of amylase by 27 isolates of various species of fungi when grown upon wheat bran. A. oryzae, Rhizopus delemar, and R. oryzae produced the largest amounts of amylase. A. oryzae was the fungus of choice because of ease of handling. In practice, fungus amylases are produced and utilized in three general ways. (1) In the amylo process, starch is solubilized by autoclaving with a trace of a mineral acid, and the mash is inoculated with a species of Rhizopus, which produces amylase abundantly, and a species of yeast. The function of the Rhizopus species is to convert the starch into ferment- able sugars, from which the yeast produces alcohol. For a description of this process see Owen (1933). (2) The fungus may be grown upon a solid substrate such as bran and the resulting moldy mass (mold bran) dried (Underkofler et al., 1946). The fresh material may be used without drying (Roberts et al., 1944). (3) Fungus amylases may be produced in submerged aerated cultures much as antibiotics are produced. The culture medium may be used directly to replace malt as a saccharifying agent. METABOLIC PRODUCTS 275 Fungus amylases are used to replace malt amylase for the saccharifica- tion of starch, Myrback (1948) is of the opinion that amylase from A. niger is an a-amylase, but it differs from a-amylase of malt in that it has a higher capacity for saccharification. For a comparison of fungus and malt amylase and the economic considerations involved, see Underkofler et al. (194G). The yield of alcohol is said to be slightly higher when fungus amylase is used in place of malt for saccharification. Fungi are the source of other enzymes of commercial interest, including pectinase and sucrase. Pectinase is used in the clarification of fruit juices. For a survey of the commercial production of fungus enzymes seeL. Wallerstein (1939). ALCOHOLIC FERMENTATION Yeasts are used almost exclusively for the commercial production of fermentation alcohol, but alcoholic fermentation is not restricted to these fungi. Pasteur (1872) observed that Penicillium glaucum, Aspergillus glaucus, and Mucor raceniosus produced alcohol under anaerobic condi- tions. Further information on alcohol production by filamentous fungi may be found in the monograph of Raistrick et al. (1931), who determined complete carbon balances for 96 species of Aspergillus, 75 species of Penicillium, 8 species of Citromyces (Penicillium) , 23 species of Fusarium and 36 miscellaneous species. The original report should be consulted for details and the quantitative methods used. All the 23 species of Fusarium studied produced alcohol. From this and other reports in the literature, it must be concluded that this property is common among species of this genus. Many species of Aspergillus and Penicillium pro- duced alcohol, as did species of other genera. Only a few of the species studied failed to produce detectable amounts of alcohol. The apparatus used in these studies is shown in Fig. 53. The concentration of alcohol which inhibits the growth of fungi varies with the species or strain. In general, yeasts are more tolerant of alcohol than the filamentous fungi. The upper limit for most yeasts is about 12 per cent alcohol, although some strains are more tolerant. The suscepti- bility to alcohol limits the alcohol concentration of naturally fermentt>? beverages. The rate of fermentation decreases as the concentration (k alcohol increases. Not all isolates of a species are equally efficient in producing alcohcu For example, eight isolates of Fusarium lini produced varying amountfe of alcohol on the same medium. The more virulent pathogens on flax produced the most alcohol (Letcher and Willaman, 1926). A correlation between sporulation and alcohol production by Aspergillus flavus was noted by Yuill (1928). In general, sporulating cultures produced less alcohol than nonsporulating cultures. The most important condition w^hich governs alcoholic fermentation 276 PHYSIOLOGY OF THE FUNGI ^ ^ r^HI sterilization Cone. Cotton wool mh: Fig. 53. Apparatus for studying the metabolic products of fungi and other micro- organisms. The apparatus consists of five units: A gasholder, P; a train for the puri- fication and sterilization of air or other gases, A-E\ the culture flask, F; a train for the quantitative absorption of carbon dioxide, H-M; an aspirator, Q, for the collection of gaseous products of metabolism other than carbon dioxide. (Redrawn from Birkin- shaw and Raistrick, Trans. Roy. Soc. (London), Ser. B, 220: 14, 1931. Published by permission of the Royal Society.) is the supply of oxygen. The relation between fermentation and anaero- bic conditions was recognized by Pasteur, who summarized his extensive investigations on fermentation as "la vie sans air." The essential feature of fermentation is anaerobic dissimilation of carbohydrates. Growth and fermentation are competitive processes, for fungi require oxygen for growth. In practice it is advantageous to carry out fermentations in the METABOLIC PRODUCTS 277 presence of some air, especially at the start. This allows some increase in the number of cells and reduces the amount of inoculum required. The amount of oxygen available to submerged mycelium or cells, unless vigorous aeration is used, is insufficient to inhibit alcoholic fermentation by certain species. Alcoholic fermentation has been studied since the time of Lavoisier. Few fields of study have been so valuable in increasing our understanding of the life processes of microorganisms. Harden (1932) has concisely reviewed the early work and theories on fermentation. The idea that yeasts as living fungi were the proximate cause of fermentation did not gain acceptance for many decades. The eminent Wohler (1839) ridiculed this idea in a lively skit, in which he declared that he had followed the entire process microscopically. Briefly, he states that the responsible organism developed from an egg and had the shape of a Beindorf distilling flask; "... diese Infusorien fressen Zucker, entleeren aus dem Darm- kanal Weingeist, und aus den Harnorganen, Kohlensaure." The enzymatic nature of alcohol fermentation was established by Buchner (1897). The enzymatic transformations involved in fermenta- tion were discussed in Chap. 7. Further information and references may be found in Summer and Somers (1947), Tauber (1949), Prescott and Dunn (1949), Meyerhof (1944, 1949), Nord and Mull (1945), and Foster (1949). The larger part of the world-wide fermentation industry is devoted to the production of ethyl alcohol. During the war year of 1945 some 600 million gallons of 95 per cent ethyl alcohol was produced in the United States alone. Less than one-third this amount was produced in 1948. Of this amount 64 per cent was produced by fermentation (Lee, 1949). While any source of fermentable sugars may be used for the production of alcohol, the more common raw materials include molasses, starch from various sources, hydrolyzed cellulose or wood sugar, and fruit juices. It is beyond the scope of this text to discuss the commercial production of industrial and beverage alcohol. For information on these subjects see Prescott and Dunn (1949). ORGANIC ACIDS Many fungi synthesize organic acids, which accumulate in the medium. These acids include oxalic, citric, succinic, fumaric, malic, lactic, itaconic, kojic, gluconic, and others. Commonly, a species may synthesize a variety of related acids. The isolates of a given species may differ widely in synthetic capacity. To obtain maximum yields, it is necessary to control nutritional and environmental factors closely. The optimum conditions for one isolate may differ from those of another isolate of the 278 PHYSIOLOGY OF THE FUNGI same species. Acid production by fungi is discussed in detail by Foster (1949), Prescott and Dunn (1949), and Wallvcr (1949). The meaning of the term fermentation has been expanded by most authors to include aerobic as well as anaerobic processes. The produc- tion of most organic acids and antibiotics Ijy fungi takes place in the presence of oxygen, and these processes are not fermentations in the restricted (anaerobic) sense of the term. Indeed, adequate aeration is one of the salient features of such processes. Aeration may be achieved by cultivating the fungi on the surface of shallow layers of medium in pans or trays ; or the fungi may be cultivated in closed tanks, which may contain as much as 15,000 gal. of medium. Aeration is provided by mechanical stirring and blowing in sterile air under pressure. The organic acids discussed in this chapter are derived from carbo- hydrates present in the medium. In general, media highly unbalanced with respect to carbohydrates are used. The balanced medium devel- oped by Steinberg for the cultivation of Aspergillus niger (Chap. 2) has a carbon-to-nitrogen ratio of 29 to 1, while the medium recommended by Currie (1917) for the production of citric acid by ^. niger has a carbon-to- nitrogen ratio of 72 to 1. A fungus first utilizes the nutrients in the unbalanced medium for the production of mycelium (growth phase). The excess carbohydrate which remains when the nitrogen is exhausted is dissimilated ("fermentation" phase). Advantage is taken of such preformed mycelium, for if the original medium is replaced by fresh medium, the mycelium continues to dissimilate carbohydrate. The replacement medium is frequently more unbalanced than the growth medium. For example, Karow and Waksman (1947) used for A. wentii a growth medium with a carbon-to-nitrogen ratio of 135 to 1, while the replacement medium had a carbon-to-nitrogen ratio of 270 to 1. Economic amounts of organic acids may accumulate in the medium because the normal use of these compounds for the synthesis of mycelium is prevented by the imposed experimental conditions. If the nitrogen supply is exhausted, no more protoplasm can be formed. The mycelium then dissimilates sugars enzymatically. Enough nutrients are supplied in replacement media to repair and maintain the enzyme systems of the fungus in a vigorously functioning state. The enzymes, other than those concerned with certain phases of carbohydrate dissimilation, are largely idle because of the lack of suitable substrates. A fungus commonly produces several organic acids at the same time. Citric and oxalic acids are produced by many isolates of A . niger, and the relative amounts of these acids may be varied by controlling the pH of the medium. In general, a highly acid medium (pH 2.0 to 3.0) favors the synthesis of citric acid, while less acid media favor the production o/ oxalic acid. METABOLIC PRODUCTS 279 Citric acid. Wehmer was the first to recognize the commercial possi- bilities of citric acid synthesis by two species of Citromyces {Penicillium). Selected isolates of Aspergillus niger appear to be used in industry, although the propertj^ of producing citric acid is common to many fungi. The following fungi have been suggested for commercial citric acid pro- duction (Von Loesecke, 1945): Citromyces pfejferianus, C. glaber, C. citricus, Aspergillus carhonarius, A. glaucus, A. clavatus, A. cinnamomeus, A.fumaricus, A. awamori, A. aureus, Penicillium arenarium, P. olivaceum, P. divaricatum, P. sanguifluus, P. glaucum, Mucor pyriformis. The production of citric acid in the United States increased from about 5 million to 26 million pounds between 1935 and 1945 (Von Loesecke. 1945). Presumably most of this was "fermentation" citric acid. At present it is beheved that most citric acid is produced by surface cultures. Citric acid is formed from many sources of carbon. Sucrose is said to be the best carbon source for the production of citric acid. There is less agreement upon the value of other sugars. Different investigators have found glucose, fructose, and maltose to vary from good to poor. In part, this is to be attributed to the use of different isolates and different experimental conditions. Beet molasses is used in industry. The suitability of this substrate is said to vary with the source and year of production (Bernhauer and Knobloch, 1941). The evaluation of carbon sources is complicated by the metallic elements they contain, especially iron and manganese. Methods of treating beet and cane molasses to remove inhibiting impurities are described by Perlman et al. (1946), Gerhardt et al. (1946), and Karow and Waksman (1947). The inhibiting effect of metallic ions on the production of citric acid from sugars is illus- trated by the data in Table 49. Table 49. The Effect of Removing Metallic Contaminants from Three Sugars, by the Process of Cationic Exchange, on the Prodlction of Citric Acid by Aspergillus niger, Wisconsin Strain 62 (Perlman et al., Arch. Biochem. 11, 1943. Published by permission of Academic Press, Inc.) Sugar used Treatment Yield* of citric acid, % Sucrose from cane Not treated 21.4 Treated 64.0 Sucrose from beet Not treated 11.3 Treated 66.8 Glucose Not treated 20.5 Treated 60.0 * Theoretical yield 123 per cent. The production of citric acid in submerged culture was tried at an early date and abandoned in favor of surface culture. However, recent litera- 280 PHYSIOLOGY OF THE FUNGI ture indicates that submerged culture may be the preferred process in the future. Average yields of 72 g. of anhydrous citric acid per 100 g. of sucrose in the medium have been obtained in the laboratory (Shu and Johnson, 1948). The formula for citric acid is given below: CH2— COOH I HO— CH— COOH CH2— COOH Citric acid Any theory of citric acid formation must take into account the following facts: Citric acid, a branched-chain compound, is synthesized from carbon sources containing from two to seven carbon atoms. Yields of citric acid may approach 90 per cent of the sugar used (Wells et al., 1936). The amount of carbon dioxide evolved is low, which suggests either reutilization or a mechanism of producing the necessary intermediates without the production of carbon dioxide. Reutilization of carbon dioxide seems the more probable, for Foster et al. (1941) showed Asper- giUus niger to utilize radioactive carbon dioxide in the synthesis of citric acid. The more probable pathway of synthesis is via the Krebs cycle (Chap. 7) and the supplementary formation of oxalacetic from pyruvic acid and carbon dioxide (Wood-Werkman reaction). Gluconic acid. A considerable number of fungi produce gluconic acid. These include Aspergillus niger (various isolates), A. fuscus, A. cinna- momeus, A. oryzae, Penicillium glabrum, P. glaucum, P. purpurogenum var. ruhrisclerotium, P. chrysogenum, P. crustaceum, and Fumago vagans. Most investigators have used selected isolates of A. niger for the produc- tion of gluconic acid. Details of laboratory and semi-pilot-plant investi- gations may be found in the papers of Wells et al. (1937), Gastrock et al. (1938), and Forges et al. (1941). Many factors influence the formation of gluconic acid. Isolates of A. niger differ in ability to synthesize this acid. Not all isolates produce the maximum amount of acid under identical conditions. Adequate aeration is necessary for the enzymatic conversion of glucose to gluconic acid. Gluconic acid is produced most abundantly when the pH of the medium is kept near 5. Calcium carbonate is used for neutralizing the gluconic acid formed. This is advantageous, for calcium gluconate is used in medicine as a source of readily assimilable calcium. Frecipitation of calcium gluconate during formation may be prevented by the addition of boric acid or borax to the culture medium in amounts vaiying up to 2,000 p. p.m. (Moyer et al., 1940). Boron compounds are added after the growth of mycelium is essentially complete. The mycelium may be used as many as thirteen times by removing the spent medium and adding METABOLIC PRODUCTS 281 fresh medium with a high glucose content but low in other nutrients (Forges et al, 1940, 1941). The production of gluconic acid appears to be a direct oxidation of glucose. The enzyme responsible for this transformation is called glucose aerodehydrogenase. This enzyme, when free from catalase, catalyzes a reaction between glucose and oxygen. Gluconic acid and hydrogen peroxide are the products formed. Glucose aerodehydrogenase was first isolated from Penicillium chrysogenum and was called notatin, or penicil- lin B, at first. Its antibiotic activity is due to liberation of hydrogen peroxide. For recent papers on this enzyme see Keilin and Hartree (1948, 1948a). Lactic acid. Various lactic acid bacteria are used in the commercial production of lactic acid. These bacteria require a complex natural medium, which makes the purification of lactic acid laborious. Many species of Phycomycetes produce lactic acid, and species of Rhizopus are noteworthy in this respect. The following fungi produce lactic acid: Rhizopus arrhizus, R. chinensis, R. elegans, R. japonicus, R. nodosus, R. oryzae, R. pseudodiinensis, R. salehrosus, R. shanghaiensis, R. stolonifer, R. tritici, Mucor rouxii, Monilia tamari, and Blastocladia pringsheimii. Most of these fungi appear to synthesize c?-lactic acid, although R. chinensis synthesizes Wactic acid (Saito, 1911). The use of R. oryzae for production of lactic acid has been intensively investigated (Lockwood et al., 1936; Ward et at., 1936, 1938). Glucose appears to be the best sugar. Nitrate nitrogen is not used by this fungus. Calcium carbonate is used in the medium to neutralize lactic acid as it is formed. Yields increase when the cultures are aerated. As much as 75 per cent of the glucose utilized is converted into lactic acid. The presence of added zinc increases mycelial growth but depresses the yield of lactic acid. The mechanism of lactic acid production by fungi is ably discussed by Foster (1949). Under anaerobic conditions, ethyl alcohol, carbon dioxide, and lactic acid are produced in equimolecular amounts. The amount of lactic acid produced under aerobic conditions increases, while the amount of alcohol decreases (Waksman and Foster, 1939). The most probable intermediate for the production of lactic acid is pyruvic acid. Itaconic acid. Aspergillus itaconicus was the first fungus reported to synthesize itaconic acid. The structural formula below shows that this unsaturated acid is related to succinic acid. CH2=C— COOH HoC— COOH Itaconic acid 282 PHYSIOLOGY OF THE FUNGI The fungi which have been tested for itaconic acid production are mainly selected isolates of A. terreus. Relatively few isolates produce sufficient itaconic acid to have commercial possibilities (Calam et al., 1939; Moyer and Coghill, 1945). Various attempts have been made to produce mutants of A. terreus by irradiating conidia with ultraviolet light (Raper et al., 1945). Less success attended these efforts than comparable treatment of conidia of Penicillium chrysogenum for obtaining mutants with enhanced penicillin production. Among the factors which affect the production of itaconic acid by isolates of .4. terreus are the composition of the medium, hydrogen-ion concentration, temperature, and aeration. Glucose and ammonium nitrate appear to be the best sources of carbon and nitrogen. The pH range in which itaconic acid accumulates is narrow and low, 1.9 to 2.3. The aluminum ion is toxic to A. terreus, but aluminum trays may be used if the concentration of magnesium ion in the medium is high. As much as 4.75 g. of magnesium sulfate heptahydrate per liter of medium may be used. It is probable that this high concentration of magnesium ion also enables the fungus to withstand low pH values (Lockwood and Ward, 1945). Fumaric acid. This unsaturated, four-carbon, dicarboxylic acid is produced by many fungi, although only a relatively few species synthe- size large amounts. With few exceptions, the fungi which synthesize fumaric acid in significant amounts are Phycomycetes. The formula for fumaric acid is given below: HOOC— CH II HC— COOH Fumaric acid The factors which affect the production of fumaric acid by Rhizopus nigricans were studied by Foster and Waksman (1939). The concentra- tion of zinc was found to be especially important. Optimum production of fumaric acid occurred in cultures receiving less zinc than that required for optimum growth. Not all isolates of R. nigricans synthesized fumaric acid in equal amounts or under the same conditions. One isolate studied by Foster and Waksman (1939a) produced fumaric acid anaerobically and aerobically, whereas another produced fumaric acid aerobically only. Various proposals have been made to explain the mechanism of fumaric acid formation. Anaerobic synthesis is thought to involve the formation of oxalacetic acid from pyruvic acid and carbon dioxide (Foster and Davis, 1948). The follo^^•ing steps would convert oxalacetic acid to fumaric acid: oxalacetate — ^ malate — > fumarate. It is probable that fumaric acid is produced aerobically from acetic acid as follows: 2 (ace- METABOLIC PRODUCTS 283 cate) —> succinate —> fumarate (Thunberg-Wieland condensation). R. nigricans produces high yields of fumaric acid from both ethyl alcohol and acetic acid, which is evidence in favor of this scheme of formation (Foster and Waksman, 1939). Other organic acids. Apparently, the first organic acid to be dis- covered as a product of fungus metabolism was oxalic acid. Many fungi in nature contain calcium oxalate crystals. This was noted as early as 1887 by De Bary. Many species of Aspergillus and Penicillium produce large amounts of oxalic acid, especially if enough alkali is present in the medium to convert the acid into an oxalate. Many species of Aspergillus which produce oxalate in the presence of a neutralizing agent also produce citric acid in acid media (Currie, 1917). For a recent discussion of oxalic acid production by fungi see Foster (1949). Various species of Aspergillus, including .4. oryzae, A. flavus, A. nidu- lans, A. giganteus, and some other fungi produce kojic acid. Kojic acid is a cyclic compound, a pyrone, and has been shown to have antibiotic properties (Morton et al., 1945). ESTERS Among the esters reported to be formed by fungi are ethyl acetate, methyl cinnamate, methyl p-methoxycinnamate, and isobutyl acetate. Various reports are in the literature concerning a "banana-oil" odor being produced by fungi, but apparently amyl acetate has not been isolated and identified as a product of fungus metabolism. Ethyl acetate is produced by Penicillium digitatum (Birkinshaw et al., 1931) and by Endoconidiophora moniliformis (Gordon, 1950). ANTIBIOTICS AND DRUGS The inhibition of one organism by another is called antagonism. The phenomenon has been known since the time of Pasteur, and the subject has been reviewed by Waksman (1947) in a book containing over 1,000 references. Antagonism occurs in nature as well as in the laboratory and is of such common occurence that it is frequently overlooked. Exam- ples are easily found by examining contaminated plates for clear areas around the contaminants. Antagonism may be due to competition for nutrients or to toxic substances. This discussion will deal wdth the toxic substances produced by fungi which inhibit fungi and bacteria. General discussion. Fungi and other organisms produce a variety of toxic substances, which include enzymes, alkaloids, toxins, simple and complex organic compounds, and inorganic compounds. Organic com- pounds produced by fungi and other organisms, especially bacteria and actinomycetes, which inhibit the life processes of microorganisms are called antibiotics. Waksman (1947) would restrict the term antibiotic 284 PHYSIOLOGY OF THE FUNGI to organic compounds produced by microorganisms which inhibit the functioning of other microorganisms. General usage of the term anti- biotic is, however, wider than this and appHes the term to those organic compounds of fairly simple structure produced by organisms which inhibit microorganisms. These substances are referred to more specifically as antibacterial, antifungal, or antiviral substances. There are no universal antibacterial or antifungal substances. Anti- biotics are specific in action. Penicillin, for example, is active against A Fig. 54. Method of assay for antibiotics. A, control culture of Penicillium notatum on agar medium; radial series of plugs cut at 6 days. B, agar-plug assay plate show- ing zones of inhibition of Staphylococcus developed after agar blocks removed from A have been incubated for 16 hr. at 37°C. (Courtesy of Raper, Alexander, and Coghill, Jour. Bad. 48: 644, 1944. Published by permission of The Williams & Wilkins Company.) many Gram-positive bacteria and only a relatively few Gram-negative organisms. The occurrence of antibiotics is probably far more widespread than suspected at present. The reason for this lies in the way in which anti- biotics are discovered. Antibiotics are detected by their inhibiting action on living organisms. A susceptible test organism is essential for the detection of an antibiotic. For obvious reasons, human pathogenic bacteria are most used for screening tests. If one desires to obtain anti- fungal substances active against pathogenic fungi, these fungi should be used as test organisms. The same principle underlies all methods for detecting antibiotic action. The test organisms are brought into contact with the products elaborated by the organism suspected of producing an antibiotic. This may be done by growing two organisms on the same Petri dish. A clear zone between METABOLIC PRODUCTS 285 the colonies indicates inhibition (Fig. 45). A second method consists in growing an organism on agar and cutting radially a series of agar plugs and placing these agar disks, which contain the antibiotic, on agar plates sown uniformly with the test organism (Raper et al., 1944). This method is illustrated in Fig. 54. Other methods of detecting antibiotics have been summarized by Waksman (1947). Fig. 55. The antibiotic effect of Streptomyces sp. on two plant pathogenic fungi, Monilinia fructicola, on the left, and Helminthosporiuin sativum, on the right. The production of antibiotic substances by fungi is common. In a screening test of over 400 species, which included over 300 wood-inhabit- ing fungi and 22 dermatophytes, somewhat over 200 species produced substances active against Staphylococcus aureus and Escherichia coli (Robbins et al., 1945). A large number of Basidiomycetes and other fungi have been tested for the presence of antibiotics by Wilkins and Harris (1944). The actinomycetes are the source of many useful anti- biotics including streptomycin, Chloromycetin, aureomycin, terramycin, and other unidentified compounds (Waksman, 1947). With the excep- tion of Phytophthora erythroseptica none of the Phycomycetes appear to have been reported as producing antibacterial substances. For a survey of Fungi Imperfecti in the role of producing antibacterial substances (against Staphylococcus aureus) and antifungal substances (against Botrytis allii), see Brian and Hemming (1947). The inhibiting effect of Streptomyces sp. on two plant pathogenic fungi is shown in Fig. 55. Many soil organisms produce antibiotics. Whether these organisms produce antibiotics in sufficient amounts to inhibit plant pathogens under natural conditions in the soil is not certain. It is known, however, that 286 PHYSIOLOGY OF THE FUNGI the incidence of certain diseases may be decreased by adding certain bacteria, actinomycetes, and fungi to soil. For references, see Grossbard (1948), Henry (1931), Waksman (1937), and Anwar (1949). The influence of various soil-inhabiting organisms in decreasing infec- tion of barley by Helminthos'porium sativum has been reported by Anwar (1949). Figure 56 illustrates some of these results. It is by no means certain that these effects were due to the antifungal substances produced by the antagonistic organisms. Fig. 56. The effects of certain soil organisms on the pathonogcnicit}^ of Helmintho- s'porium sativum on barley. Seedlings grown at 80°F. in steamed soil infested with: A, no organisms; B, H. sativum and Bacillus subtilis: C, H. sativum and Penicillium sp. ; D, H. sativum and Trichoderma lignorum: E, H. sativum. (Courtesy of Anwar, Phytopathology 39: 1011, 1949.) The situation in soil is very complicated. Basic antibiotics such as streptomycin are adsorbed on clay; acidic antibiotics like clavacin are apparently held less firmly. Gottlieb and Siminoff (1950) are of the opinion that competition is more of a factor than antibiotic action as the cause of one organism inhibiting another in the soil. Thus, either As-per- gillus clavatus or Streptomyces griseus inhibits the growth of Bacillus suhtilis in soil. No difference was noted between a strain of S. griseus which produced streptomycin and one which did not. Schatz and Hazen (1948) reported that 124 of the 243 soil Actiyiomyces METABOLIC PRODUCTS 287 tested were antagonistic to four test human pathogens, Candida albicans, Cryptococciis neoformans, Trichophyton gypseum, and T. rubrum. Table 50. Antibiotics Produced by Soil-inhabiting Actinomycetes and Fungi (Brian, Cheni. Industry 1949. Published by permission of the Society of Chemical Industry.) Organism Streptomyces griseus Nocardia gardneri Actinomyces lavendulae Proactinomyces cyaneus Streptomyces venezuelae Aspergillus jlavus A. terreus Fusarium orthoceras Penicillium brevi-compactum P. chrysogenum P. griseofulvum P. janczewiskii P. patulum Trichoderma viride Antibiotic Grisein Actidione Streptomycin Proactinomycin Streptothricin Litmocidin Chloromycetin Aspergillic acid Citrinin Enniatin B Mycophenolic acid Penicillin Griseofulvin Griseofulvin Patulin Gliotoxin Viridin Properties Antibacterial (Gram positive and negative) ; antirickettsial ; not antifungal Not antibacterial; antifungal Antibacterial (Gram positive and negative and acid fast) Antibacterial (Gram positive) Antibacterial (Gram positive) ; antifungal Antibacterial (Gram positive and negative) Antibacterial (Gram positive and negative); not antifungal; anti- rickettsial Antibacterial (Gram positive and negative); antifungal Antibacterial (Gram positive and negative); antifungal Antibacterial (Gram positive and acid fast) ; not antifungal Antibacterial (Gram positive and negative); antifungal Antibacterial (Gram positive) ; not antifungal Not antibacterial; antifungal Not antibacterial; antifungal Antibacterial (Gram positive and negative) ; antifungal Antibacterial (Gram positive and negative and acid fast) ; anti- fungal Not antibacterial; antifungal A list of antibiotics produced by some soil-inhabiting actinomycetes and fungi is given in Table 50. Note that some organisms produce more than one antibiotic and that the same antibiotic substance may be pro- duced by more than one species. Organisms differ in susceptibility to antibiotics. This range of effectiveness is frequently called the antibioHc spectrum. Thus, Penicillum luteum-purpurogenum is some 12 thousand times as sensitive to gliotoxin as to streptomycin. Not all fungi are equally inhibited by the same concentration of an antibiotic; some 11 288 PHYSIOLOGY OF THE FUNGI times as much clavacin is required to inhibit the growth of Aspergillus clavatus as Trichophyton mentagrophytes (Reilly et al., 1945). Fungi produce substances which are capable of inactivating certain plant viruses. The Basidiomycetes are especially noteworthy in this respect (Utech and Johnson, 1950). Extracts of Trichotheciiim roseum reduce infectivity of southern bean mosaic, tobacco mosaic, and tobacco necrosis viruses (Gupta and Price, 1950). These authors believe that this reduced infectivity is due to increased resistance of the host. There is no evidence which indicates that any of the known antibiotics are involved in the destruction of plant viruses. However, antibiotics are known which are effective against virus diseases in man. Preliminary studies indicate that certain antibiotics may be used to control fungi which cause plant diseases. Actidione has been reported by Vaughn et al. (1949) to control powdery mildew on beans and roses. Actidione was toxic to young rose leaves at a concentration of 2.5 p.p.m. but less toxic to bean plants. Laboratory tests indicated that actidione is a fungistatic substance for a considerable number of plant pathogenic fungi, including Sclerotinia fructicola, Cladosporium cucumerinum, and Colletotrichum lagenarium. Further data on the effect of actidione on plant pathogenic fungi are reported by Whiffin (1950). The protective action of an antibiotic obtained from an unidentified species of Streptomyces against Venturia inaequalis on apple has been reported by Leben and Keitt (1949). This antibiotic has been named antimycin. Penicillin has been used successfully, to a limited extent, in controlling necrosis of giant cactus, caused by Erwinia carnegieana (Boyle, 1949). Injections of penicillin into the necrotic tissue apparently diffused through the plant tissues for some distance, killing the bacteria. This is one of the few cases in which an antibiotic has been used successfully in thera- peutic treatment of plant disease. The principal use of antibiotics is to control disease in man and animals. Only a relatively few antibiotics are useful for this purpose. In addition to killing or inhibiting pathogenic organisms, an antibiotic, to be useful in medicine, must be relatively nontoxic to the host. Some of the older and more useful antibiotics used in medicine will be discussed in greater detail on the following pages. Penicillin. This antibiotic drug is produced in industry by selected isolates or mutants of Penicillium chrysogcnum and P. notatum. The original isolate of Fleming produced from 2 to 4 units of penicillin per milliliter of culture filtrate. P. chrysogenum Q-17Q has produced in excess of 1,000 units per milliliter. The synthesis of penicillin is not limited to species of the P. chrysogenum-notatum group but includes cer- tain species of Aspergillus belonging to the A. flavus group. A few fungi METABOLIC PRODUCTS 289 belonging to other genera also produce penicillin. Sterile culture condi- tions must be maintained at all times, as penicillin is rapidly destroyed by the enzyme, penicillinase, which is excreted by many bacteria. Peni- cillin is extracted from the "fermentation" liquid, or penicillin beer, either by extraction at pH 2.0 to 2.5 with water-immiscible solvents such S R— — CO— NH— CH- 0=0— -CH C— (CH3)2 -N CH— COOH Type formula of the penicillins as amyl acetate, or by adsorption on activated carbon. The extraction must be carried out quickly from acid solutions, owing to the instability of penicillin under these conditions. Penicillin forms crystalline salts with the alkali metals. The sodium salt is usually produced. Table 51. Names and American and British-type Designations of Four Naturally Occurring Penicillins R Name Type American British Benzylpenicillin A^-Pentenylpenicillin p-HydroxybenzylpenicillLn n-Heptylpenicillin G F X K II CHs- CHo— CH=CH— CHo— HOC6H4CH2— (j-H.3(0-H.'>)5 — C-H.'> — I III IV Penicillin, as produced by P. chrysogenum, is a mixture of related com- pounds. The ratios among the various penicillins depends upon the isolate and conditions used. By the use of suitable precursors the yield of the desired compound, penicillin G, is greatly increased. This is desirable because penicillin G salts crystallize well and are most useful in medicine. The type formula for the penicillins is shown below. The precursors used for the production of penicillin G are related to phenyl- acetic acid. Many other penicillins are known, some of which are pro- duced only in the presence of precursors which do not occur in nature (Behrens, 1949). The growth of the penicillin industry in the United States is shown by the production figures in Table 52. Penicillin is chiefly active against Gram-positive bacteria. A few important Gram-negative pathogens, including Neisseria gonorrhoeae and Treponema pallidum, are controlled by penicillin. Penicillin is not active against acid-fast bacteria or fungi. Many bacteria may become resist- ant, or fast, to penicillin. Whether natural selection or mutation or both are involved in this phenomenon is uncertain. The morphology and 290 PHYSIOLOGY OF THE FUNGI physiology of penicillin-fast bacteria vao^y be abnormal (Bellamy and Klimek 1948). Penicillin is most active against young cells, in that it inhibits the process of cell division. For papers on the mechanism of penicillin action see Cavallito et al. (1945), Chain and Diithie (1945); Bailey and Cavallito (1948) ; and a series of papers by Pratt and Dufrenoy (1949). Table 52. The Production of Penicillin in the United States for the Years 1943 TO 1948 A unit of penicillin is 0.6 ^g. (Coghill and Koch, Chem. Eng. Neivs 23, 1045; Lee, Ind. Eng. Chem. 41, 1949. Published by permission of the American Chemical Society.) Year Billions of Units 1943 21 1944 1,633 1945 6,852 1946 25,809 1947 41,426 1948 95,855 Further details may be found in the following selected references. For a concise authoritative account of all phases of penicillin, see Foster (1949). The medical aspects of penicillin therapy are discussed by Fleming (1949). The chemistry of penicillin is covered in the monograph edited by Clarke et al. (1949). The early history of penicillin is pre- sented by Chain and Florey (1944) and Waksman (1947). Streptomycin. This antibiotic was discovered in Waksman's labora- tory in 1943, and three years later, commercial production of this drug began. Streptomycin is synthesized by some isolates of Streptomyces griseus. The techniques used in industry resemble those used for the production of penicillin in submerged aerated culture. Streptomycin is adsorbed on activated carbon as the first step in isolation and purifica- tion. In contrast to penicillin, streptomycin is a basic compound. The production of streptomycin in the United States increased from 1,175 billion units in 1946 to 37,710 billion units in 1948 (Lee, 1949). The chemistry of streptomycin is reviewed by Lemieux and Wolfrom (1948). Streptomycin is mainly active against Gram-negative bacteria and certain acid-fast organisms, including Mycohacterium tuberculosis. This drug controls many pathogens which are unaffected by penicillin. Organ- isms exposed to streptomycin frequently become fast. Indeed, some bacteria have been reported to become dependent upon the drug. The composition of the medium influences streptomycin production. Soybean meal appears to be a suitable source of nitrogen for commercial production (Rake and Donovick, 1946). The influence of carbon and nitrogen sources in synthetic media has been studied by Dulaney (1948, 1949). These results may be summarized as follows: Glucose and man- METABOLIC PRODUCTS 291 nose are the best hexoses; maltose is the best disaccharide ; starch and its degradation product dextrin are good carbon sources. Streptomyces griseus does not utihze nitrate nitrogen. L-ProHne is the most favorable single amino acid, but addition of this amino acid to other sources of nitrogen does not increase yields (see also Thornberry and Anderson, 1948). A popular account of the development of streptomycin is to be found in Epstein and Williams (1946). Aureomycin. This antibiotic is produced by Streptomyces aureofaciens. Aureomycin has been prepared in pure crystalline form (Duggar, 1948). In addition to being effective against many Gram-positive and Gram- negative bacteria, aureomycin is also active against certain rickettsial and viral agents. Preliminary reports on the use of aureomycin for treating Rocky Mountain spotted fever have been favorable (Schoenbach et al., 1948). Aureomycin appears to be of great clinical value in treating lymphogranuloma venereum and granuloma inguinale, two virus diseases of man (Wright et al, 1948). Chloromycetin. This antibiotic was discovered independently in two laboratories (Ehrlich et al., 1947, and Carter et al., 1948). It is produced by Strepto77iyces venezuelae and is the first useful antibiotic to be produced synthetically on a commercial scale. Chloromycetin is active against certain Gram-positive and Gram-negative bacteria, acid-fast bacteria, and Rickettsia (Smith et al., 1948). It has been reported that this anti- biotic has been used successfully to treat epidemic typhus (Payne and Knaudt, 1948). Chloromycetin contains non-ionic chlorine and a nitro group, two unusual features in compounds of biological origin. The formula is given below : O H NHCOCHClo N— ^C— C— CH2OH O HO H Chloromycetin (Chloramphenicol) Ergot. Among the alkaloids produced by fungi, only those obtained from the sclerotia of Claviceps purpurea appear to be used in medicine. Seven related alkaloids have been isolated from ergot. These alkaloids have different pharmacological properties. The most useful alkaloid, ergonovine, is isolated from the others before use. Certain undesirable effects of the other alkaloids are avoided by this procedure. Ergonovine, and formerly a mixture of ergot alkaloids, is used to stimulate uterine contraction. At present there is no satisfactory synthetic substitute for ergonovine (Mass, 1950). In 1941, 571,000 lb. and in 1944, 85,000 lb. of ergot sclerotia were imported into the United States. If the sclerotia are consumed in large 292 PHYSIOLOGY OF THE FUNGI enough quantities by man or animals, they cause ergotism, a disease also known as St. Anthony's fire. C. purpurea has been cultured under laboratory conditions but forms neither sclerotia nor alkaloids under these conditions (Michener and Snell, 1950). Apparently alkaloids are formed only in the sclerotia. Ergotamine, when added to mycelial cultures of C. purpurea, was largely destroyed. TOXINS Numerous toxic substances are produced by fungi in nature, and their effects on man and animals are varied. The most severe toxins are produced by some of the Agaricaceae, particularly by species of Amanita. It is not known whether the toxins are present in the mycelium of these species as well as in the fruit bodies. A few of the outstanding examples of fungus toxins will be discussed briefly. Amanita toxin (phalloidin) is stable to heat and drying and to the action of digestive juices. The great majority of the deaths due to mushroom poisoning are caused by Amanita phalloides, A. virosa, and A. verna, which contain amanita toxin. The action of this toxin is slow, the symp- toms being delayed for 6 to 15 hr. after the mushrooms are eaten. By this time the toxin has been absorbed, and the patient seldom responds to treatment. No antidote for this toxin is known. The mortality rate is high, varying from 60 to 100 per cent (Fischer, 1918). In addition to the three species of Amanita mentioned above, the same or a similar toxin is present in A. spreta, A. porphyria, A. strohiliformis, A. radicata, and A. chlorinosma. Hygrophorus conicus and Pholiota autumnalis produce similar symptoms and may contain this toxin (Krieger, 1936). About 1 g. of pure crystalline toxin can be extracted from 40 kg. of A. phalloides fruit bodies. The toxic dose for white mice is 50 jug; death results in from 1 to 2 days. Chemically, phalloidin is a polypeptide containing six amino-acid residues. Wieland and Witkop (1940) report that phalloidin, on hydrolysis wdth sulfuric acid, yields 1 mole each of Z-Q!-oxytryptophane and cysteine, and 2 moles each of ^-hydroxy proline b (not found in protein digests) and /-alanine. Kuhn et al. (1939) found methionine in addition to cysteine (ratio 1 to 5) in phalloidin. Among the antibiotics, gramicidin and tyrocidine are polypeptides which contain "unnatural" amino acids and are toxic when injected into experimental animals. Muscarin is the principal toxic agent present in A. muscaria. It is a quick-acting toxin, producing symptoms within 1 to 6 hr. after being consumed. The patient usually responds well to treatment and recovery is rapid, although death may occur. Atropin is an antidote for muscarin which is closely related to choline. Muscarin has also been demonstrated in A. pantherina, Russula emetica. Boletus luridus, and B. satanas. A METABOLIC PRODUCTS 293 similar or the same toxin is present in Clitocyhe illudens, Inocyhe infelix, I. infida, Lactarius torminosus, and B. miniato-olivaceus var. sensibilis (Krieger, 1936; Wolf and Wolf, 1947). HelveUic acid is known to be present only in Gyromitra esculenta. Ap- parently there is considerable variability in the reaction of individuals who eat this fungus. Many people have eaten it with no ill effects, although a number of cases of poisoning and even a few deaths have been reported. Helvellic acid is soluble in hot water. Its toxic action is due to its blood-dissolving power. While a number of other toxic substances have been detected in the fleshy fungi, the exact identity of most of them is not known. For example, species of Paneolus may cause temporary paralysis or intoxica- tion similar to alcoholic intoxication. Some species of Amanita have been reported to contain other toxins in addition to those discussed above (Fischer, 1918). The alkaloids produced by Claviceps purpurea and C. paspali are toxic to man and animals if consumed in large enough quantities. The specific alkaloids produced were mentioned previously. The production of toxins is also common in the genus Fusarium. Gihberella zeae, the cause of scab of small grains, produces toxic substances which are poisonous to livestock fed on scabby grain (Christensen and Kernkamp, 1936). Numerous other fungi which cause plant diseases are known to produce toxic substances which kill the host or modify its activity (see Chap. 17). PIGMENTS Colored compounds produced by fungi and other organisms are called pigments. In the fungi, some pigments accumulate in the mycelium and spores, while others diffuse into the culture medium. The pigments produced by a fungus are in part determined by genetic factors and in part by the environment. Mycelium, fruit bodies, and spores may be pigmented, or in some species the pigment is confined to the spores. Among the fleshy fungi, brown is one of the most common colors of fruit bodies, with yellow, orange, and red being somewhat less common. Often a number of pigments are obviously present. Few fungi are green. Yet, Chlorosplenium aeruginosum produces a green pigment, sylindein (Wolf and Wolf, 1947), which stains the wood in which it grows. Blue- stain fungi (CeratostomeUa spp.) excrete blue pigments into wood. Some species of Boletus produce a blue or bluish-green pigment when bruised or wounded. Tricholoma personatum and Laccaria amythestina are among the mushrooms producing purple or violet pigments. It is said that the red-orange pigment of the fruit bodies of Echinodontium tinctorium, the Indian paint fungus, was used by the Indians as make-up. Few of the 294 PHYSIOLOGY OF THE FUNGI larger fruit bodies of the fungi are entirely black, although this is a com- mon color for perithecia, pycnidia, and spores. Among the nutritional factors which modify the production of pigments by fungi in culture, the micro essential elements, the carbon and nitrogen sources, the initial pH of the medium, and the temperature are important. Perhaps the first of these factors to be studied was the effect of iron, cop- per, zinc, and other micro elements upon the spore color of Aspergillus niger. Copper seems to play an outstanding role in the production of dark spores by this fungus (Mulder, 1939), but low concentrations of other micro essential elements also affect spore color of this fungus. The influence of iron, copper, and zinc on the pigmentation of mycelium and spores, and the production of soluble pigments by certain species was studied by Metz (1930). The investigation of the chemical structure of fungus pigments has formed an essential part of a comprehensive study of the products of fungus metabolism at the University of London. The citations in this paragraph will give the reader entry into this excellent work. Many fungi produce anthraquinone pigments. Helminthosporium gramineum stores in its mycelium two pigments (helminthosporin and hydroxyisohel- minthosporin), which may account for 30 per cent of the dry weight of the mycelium. Helminthosporin is 2-methyl-4,5,8-trihydroxyanthra- quinone (Charles et at., 1933). H. cynodontis and H. euchlaenae form cynodontin, l,4,5,8-tetrahydroxy-2-methylanthraquinone, which is closely related to helminthosporin (Raistrick et at., 1933). Some 12 anthra- quinone pigments are produced by fungi (Howard and Raistrick, 1949). Xanthone pigments are produced by H. ravenelUi and H. turcicum (Rai- strick et at., 1936). The production of anthraquinone pigments is not restricted to species of Helminthosporium, for Penicillium islandicum synthesizes chrysophanic acid, 4,5-dihydroxy-2-methylanthraquinone (Howard and Raistrick, 1950). In general, the production of these and other pigments is modified by the cultural conditions used. The produc- tion of helminthosporin by H. gramineum was increased when nitrate or organic sources of nitrogen were used. Ammonium nitrogen was not favorable for pigment production. More pigment was produced when the initial pH was 8 than in more acid media. Many of the water-soluble pigments produced by fungi are indicators. P. phoeniceum and P. ruhrum produce such an indicator pigment, phoe- nicine (2,2'-dihydroxy-4,4'-ditoluquinone). The color changes of this indicator are from yellow to red in the pH range of 1.8 to 3.4 and from red to violet in the range 5.4 to 6.4. As much as 2 g. of this pigment is produced by P. ruhrum per liter of medium (Curtin et al., 1940). The functions of fungus pigments are not well understood. It is known that certain of these pigments are enzyme inhibitors. Others, like METABOLIC PRODUCTS 295 citrinin, are antibiotics. The physiological activity of solanione, a purple pigment produced by Fusarium solani, decreases growth and the efficiency of fat formation by F. lini (Weiss and Nord, 1949). Solanione is a 1,4-naphthoquinone, and this activity is in accord with the effects of other compounds of this series. F. graminearum synthesizes an orange-red pigment, rubofusarin, Avhich is a xanthone (Ashley et at., 1937). Rubo- fusarin was found to stimulate growth and to inhibit the enzymatic dehydrogenation of isopropyl alcohol by F. lini (Sciarini et al., 1943). It is suggestive that pigment production frequently occurs near the time of maximum development of mycelium. Perhaps pigments influence sporulation in some way. Carotene is produced by Mucor hiemalis and Phycomyces blakesleeanus, and probably by Mucor mucedo, Pilairia anomala, and Dicranophora fulva (Schopfer, 1935). The amount of carotene produced by Phycomyces blakesleeanus was increased by increasing the concentration of asparagine in the medium. The plus strain of this fungus synthesized more carotene than the minus strain. Carotene occurs in nature as three isomeric compounds, all of w'hich may be converted into vitamin A. The carotene found in M. hiemalis and P. blakesleeanus is ;S-carotene. Emerson and Fox (1940) found 7-carotene to be associated with the male gametangia of a certain species of Allomyces. Apparently carotene is also common among the yellow or orange Ascomycetes and Basidiomycetes. SUMMARY The saprophytic fungi play an important, if not indispensable, part in the degradation and decay of plant and animal residues. The most important product of fungus metabolism in nature is carbon dioxide. Humus is in part a result of the activities of soil-inhabiting fungi. Various species of fungi have been used for the preparation of food and beverages. Fungi may be used to increase the world's supply of food. Yeasts and other fungi are able to convert w-aste carbohydrate and inorganic nitrogen compounds into protein, fats, and vitamins. Yeast protein, because of its content of essential amino acids, has value as a protein supplement. Alcoholic fermentation is not restricted to the yeasts, although these fungi are used almost exclusively in industrj^ for this purpose. The production of alcohol requires anaerobic or partially anaerobic conditions. Fungi may be used to produce organic acids, of which citric acid is the most important commercially. In general, a medium high in carbo- hydrate and low in nitrogen favors the production of organic acids, which are synthesized in quantity only after growth is essentially complete. In nature one organism may be antagonistic to another because of the competition for nutrients or because of the production of antibiotics. 296 PHYSIOLOGY OF THE FUNGI Relatively feAV of the antibiotics known to be produced meet the pre- requisite of being nontoxic to man, but some of those which do are enor- mously important. Ergonovine, one of the ergot alkaloids obtained from the sclerotia of Claviceps purpurea, is a useful drug for which no synthetic substitute is available. A number of fleshy fungi produce toxins, some of which are deadly to man if consumed in sufficient quantity. The toxins vary in chemical nature, in severity, and in the symptoms they produce. Pigments are assumed to serve a definite function in fungi, at least in some instances. Some pigments are known to be antibiotics; others such as carotene are provitamins; but in general the functions of the fungus pigments remain unknown. REFERENCES Anwar, A. A.: Factors affecting the survival of Helrninthosporium satirum and Fusarium lini in soil, Phytopathologij 39: 1005-1019, 1949. Ashley, J. N., B. C. Hobbs, and H. Raistrick: Studies in the biochemistry of micro-organisms. LIII. The crystalHne colouring matters of Fusarium cul- moruyn (W. G. Smith) Sacc. and related forms, Biochem. Jour. 31 : 385-397, 1937. Bailey, J. H., and C. J. Cavallito: The reversal of antibiotic action, Joiir. Bad. 55: 175-182, 1948. Behrens, O. K. : Biosynthesis of penicillins in The Chemistry of Penicillin, Prince- ton University Press, Princeton, N.J., 1949. Bellamy, W. D., and J. W. Klimek: Some properties of penicillin-resistant Staphy- lococci, Jour. Bad. 55: 153-160, 1948. Bernhauer, K., and H. Knob loch: Ueber die Saiirebildung aus Zucker durch Aspergillus niger. XI. ]\Iitteilung. Factoren der Citronensaureanhaufung 2, Biochem. Zeit. 309: 151-178, 1941. BiRKiNSHAW, H., J. H. V. Charles, and H. Raistrick: Studies in the biochemistry of micro-organisms. XVIII. Biochemical characteristics of species of Peni- cillium responsible for the rot of citrus fruits, Trans. Roy. Soc. (London), Ser. B, 220:355-362, 1931. Birkinshaw, J. H., and H. Raistrick: Studies in the biochemistry of micro- organisms. II. Quantitative methods and technique of investigation of the products of metabolism of micro-organisms, Trans. Roy. Soc. (London), Ser. B, 220: 11-26, 1931. Block, R. J., and D. Rolling: The amino acids yielded by various yeasts after hydrolysis of the fat-free material. A comparative investigation. Arch. Biochem. 7: 313-321, 1945. *Boyle, a. M.: Further studies of the bacterial necrosis of the giant cactus, Phyto- pathology 39 : 1029-1052, 1949. Bray, C. W. : Vitamin B deficiency in infants: its possibility, prevalence and prophyl- axis, Trans. Roy. Soc. Trap. Med. Hyg. 22 : 9-42, 1928. Brefeld: O.: Untersuchungen aus dem Gesamtgebeit der Mykologie, Vol. XIV, Kommissions-Verlag von Heinrich Schoningh, Muenster, 1908. Brian, P. W.: The production of antiobiotics by soil microorganisms, Chem. & Industry, 1949, 391-393. Brian, P. W., and H. G. Hemmimg: Production of antifungal and antibacterial METABOLIC PRODUCTS 297 substances by fungi : Preliminary examination of 1G6 strains of Fungi Imperfecti, Jour. Gen. Microbiol. 1: 158-167, 1947. Brink, N. G., F. W. Holly, C. H. Shunk, E. W. Peel, J. J. Cahill, and K. FoLKfiRs: Vitamin B12. IX. l-a/p/ia-D-ribofuranosido-.5,6-dimethylimida- zole, a degradation product of vitamin B12, Jour. Am. Chem. Soc. 72, 1866, 1950. BucHNER, E. : Alcoholische Garung ohne Hefezellen, Ber. d. deut. chem. Ges. 30: 117-124, 1897. BuLLER, A. H. R. : The fungus lore of the Greeks and Romans, Trans. Brit. Mycol. Soc. 5:21-66, 1914. BuRKHOLDER, P. R. I Influence of some environmental factors upon the production of riboflavin by a yeast, Arch. Biochem. 3 :121-129, 1943. Calam, C. T., a. E. Oxford, and H. Raistrick: Studies in the biochemistry of micro-organisms. LXIII. Itaconic acid, a metabolic product of a strain of Aspergillus terreus Thom., Biochem. Jour. 33: 1488-1495, 1939. Carter, H. E, D. Gottlieb, and H. W. Anderson: Chloromycetin and strepto- thricin, Science 107: 113, 1948. *Cavallito, C. J., J. H. Bailey, T. H. Haskell, T. H. McCormick, and W. F. Warner: The inactivation of antibacterial agents and their mechanism of action. Jour. Bad. 50: 61-69, 1945. Chain, E., and E. S. Duthie: Bacteriocidal and bacteriolytic action of penicillin on the Staphylococcus, Lancet 248 : 652-657, 1945. Chain, E., and H. W. Florey: Penicillin, Endeavour 3: 3-14, 1944. Charles J. H. V., H. Raistrick, R. Robinson, and A. R. Todd: Studies in the bio- chemistry of micro-organisms. XXVIII. Helminthosporin and hydroxy/so- helminthosporin, metabolic products of the plant pathogen Helminthosporium gramineum Rabenhorst., Biochem. Jour. 27: 499-511, 1933. Christensen, J. J., and H. C. H. Kernkamp: Studies on the toxicity of blighted barley to swine, Minn. Agr. Expt. Sta. Tech. Bull. 113, 1936. Clarke, H. T., J. R. Johnson, and R. Robinson (Editors) : The Chemistry of Penicillin, Princeton University Press, Princeton, N.J., 1949. CoGHiLL, R. D., and R. S. Koch: Penicillin, a wartime accomplishment, Chem. Eng. Neivs 23: 2310-2316, 1945. CuRRiE, J. N.: The citric acid fermentation of Aspergillus niger, Jour. Biol. Chem. 31: 15-37, 1917. CuRTiN, T., G. Fitzgerald, and J. Reilly: Production of phoenicine on synthetic media. 1. Penicillium phoeniceum van Beyma. 2. Penicillium ruhrum Grassbergcr-Stoll., Biochem. Jour. 34: 1605-1610, 1940. DuGGAR, B. M.: Mushroom Growing, Orange Judd Publishing Co., Inc., New York, 1915. DuGGAR, B. M.: Aureomycin: a product of the continuing search for new antibiotics, Ann. N.Y. Acad. Sci. 51: 177-181, 1948. Dulaney, E. L. : Observations on Streptomyces griseus. III. Nitrogen sources for growth and streptomycin production. Jour. Bact. 56: 305-313, 1948. Dulaney, E. L. : Observations on Streptomyces griseus. II. Carbon sources for growth and streptomycin production, Mycologia 41 : 1-10, 1949. Ehrlich, J., Q. R. Bantz, R. M. Smith, D. A. Joslyn, and P. Bunkholder: Chlo- romycetin, a new antibiotic from a soil Actinomycete, Science 106: 417, 1947. Emerson, R., and D. L. Fox: Gamma-carotene in the sexual phase of the aquatic fungus Allomyces, Proc. Roy. Soc. (London), Ser. B, 128: 275-293, 1940. ♦Epstein, S., and B. Williams: Miracles from Microbes, the Road to Streptomycin, Rutgers University Press, New Brunswick, 1946. Erb, N. M., R. T. Wisthoff, and W. L. Jacobs: Factors affecting the production oi 298 PHYSIOLOGY OF THE FUNGI amylase by Aspergillus niger, strain NRRL 337, when grown in submerged culture, Jour. Bad. 55: 813-821, 1948. Fischer, O. E.: Mushroom poisoning in C. H. Kauffman, The Agaricaceae of Michi- gan, Vol. I, Publication 26, Biological Series of the Michigan Geological and Biological Survey, Lansing, 1918. FiTZPATRicK, W. H., W. B. EssELEN, and E. Weir: Composition and nutritive value of mushroom protein, Jour. Atn. Dietet. Assoc. 22 : 318-322, 1946. Fleming, A.: Penicillin, Its Practical Application, The Blakiston Company, Phila- delphia, 1949. Foster, J. W.: Riboflavin, British patent 593,027, Oct. 7, 1947. Chem. Abs. 42: 1710, 1948. Foster, J. W.: Chemical Activities of Fungi, Academic Press, Inc., New York, 1949. Foster, J. W., S. F. Carson, S. Ruben, and M. D. Kamen: Radioactive carbon as an indicator of carbon dioxide utilization. VII. The assimilation of carbon dioxide by molds, Proc. Natl. Acad. Sci. U.S. 27: 590-596, 1941. Foster, J. W., and J. B. Davis: Anaerobic formation of fumaric acid by the mold Rhizopus nigricans, Jour. Bad. 56: 329-338, 19-8. Foster, J. W., and S. A. Waksman: The specific effect of zinc and other heavy metals on growth and fumaric acid production by Rhizopus, Jour. Bad. 37: 599-617, 1939. Foster, J. W., and S. A. Waksman: The production of fumaric acid by molds belonging to the genus Rhizopus, Jour. Am. Chem. Sac. 61 : 127-135, 1939a. Gastrock, E. a., N. Porges, P. A. Wells, and A. J. Moyer: Gluconic acid produc- tion on pilot-plant scale. Effect of variables on production by submerged mold growths, Ind. Eng. Chem. 30: 782-789, 1938. Gerhardt, p., W. W. Dorrell, and I. L. Baldwin: Citric acid fermentation of beet molasses, Jour. Bad. 52 : 555-564, 1946. GoRciCA, H. J., and H. Levine: Vitamin Bi assimilation by yeast, U.S. patent 2,295,036, Sept. 8, 1942. Gordon, M. A. : The physiology of a blue stain mold with special reference to pro- duction of ethyl acetate, Mycyologia 42 : 167-185, 1950. Gottlieb, D., and P. Siminoff: The role of antibiotics in soil. Phytopathology 40: 11, 1950. Grossbard, E.: Production of an antibiotic substance on wheat straw and other organic materials in the soil, Nature 161: 614-615, 1948. Gupta, B. M., and W. C. Price: Production of plant virus inhibitors by fungi. Phytopathology 40 : 642-652, 1950. Hao, L. C, E. I. FuLMER, and L. A. Underkofler: Fungal amylases as saccharify- ing agents in the alcoholic fermentation of corn, Ind. Eng. Chem. 35: 814-818, 1943. Harden, A.: Alcoholic Fermentation, 4th ed., Longmans, Roberts and Green, London, 1932. Harris, E. E.. M. L. Hannan, and R. R. Marquardt: Production of food yeast from wood hydrolysates, Ind. Eng. Chem. 40 : 2068-2072, 1948. Hendlin, D., and M. L. Ruger: The effect of cobalt in the microbial synthesis of LLD-active substances. Science 111 : 541-542, 1950. Henry, A. W.: The natural microflora of the soil in relation to the foot-rot problem of wheat. Can. Jour. Research 4 : 69-77, 1931. Hesse, A.: Industrial biosyntheses. I. Fats, Advances in Enzymol. 9: 653-704, 1949. HiCKEY, R. J.: The inactivation of iron by 2,2'-bipyridine and its effect on ribo- flavin synthesis by Clostridium acetobutylicum. Arch. Biochem. 8: 439-447, 1945. METABOLIC PRODUCTS 299 ♦Howard, B. H., and H. Raistrick: Studies in the biochemistry of micro-organisms. 80. The colouring matters of PeniciUium islandicum Sopp. Part 1. 1,4,5-tri- hydroxy-2-methylanthraquinone, Biochem. Jour. 44: 227-233, 1949. Howard, B. H., and H. Raistrick: Studies in the biochemistry of micro-organisms. 81. The colouring matters of PeniciUium islandicum Sopp. Part 2. Chryso- phanic acid, 4,5-dihydroxy-2-methylanthraquinone, Biochem. Jour. 46:49-53, 1950. HuMFiELD, H.: The production of mushroom mycelium {Agaricus campestris) in submerged culture. Science 107 : 373, 1948. Karow, E. O., and S. A. Waksman: The production of citric acid in submerged culture, Ind. Eng. Chem. 39 : 821-825, 1947. Keilin, D., and E. F. Hartree: Properties of glucose oxidase (Notatin), Biochem. Jour. 42 : 221-229, 1948. Keilin, D., and E. F. Hartree: The use of glucose oxidase (Notatin) for the deter- mination of glucose in biological material, and for the study of glucose-producing systems by manometric methods, Biochem. Jour. 42 : 230-238, 1948a. Krieger, L. C. C: The Mushroom Handbook, The Macmillan Company, New York 193G. KuHN, R., L. BiRKHOFER, and F. W. Quackenbush: Jodometrische Titration von SH-gruppen: Mikromethode zur Bestimmung von Cystein und Methionin in Proteinen, Ber. d. deut. chem. Ges. 72 : 407-416, 1939. Leach, J. G.: Insect Transmission of Plant Diseases, McGraw-Hill Book Company, Inc., New York, 1940. *Leben, C, and G. W. Keitt: Laboratory and greenhouse studies of antimycin preparations as protectant fungicides. Phytopathology 39: 529-540, 1949. Lee, S. B.: Fermentation, Ind. Eng. Chem. 41 : 1838-1879, 1949. Le Mense, E. H., J. CoRMAN, J. M. vanLanen, and A. F. Langlykke: Production of mold amylase in submerged culture. Jour. Bact. 54: 149-159, 1947. Lemiei-x, R. U., and M. L. Wolfrom: The chemistry of streptomycin, Advances in Carbohydrate Chem. 3 : 337-384, 1948. Letcher, H., and J. J. Willaman: Biochemistry of plant diseases. VIII. Alcoholic fermentation of Fusarium lini, Phytopathology 16: 941-949, 1926. Levine, H., J. E. Oyaas, L. Wanerman, J. C. Hoogerheide, and R. M. Stern: Riboflavin production by Candida yeasts, Ind. Eng. Chem. 41 : 1665-1668, 1949. LiLLiE, R. J., C. A. Denton, and H. R. Bird: Relation of vitamin B12 to the growth factor present in cow manure, Jour. Biol. Chem. 176: 1477-1478, 1948. Linder, p.: Das Problem der biologischen Fettbildung und Fettgewinnung, Zeit. angeiv. Chem. 35: 110-114, 1922. Lintzel, W.: Ueber den Nahrwert des Eiweisses der Speisepilze, Biochem. Zeit. 308: 413-419, 1941. LocKWOOD, L. B.: A study of the physiology of PeniciUium javanicum van Beijma with special reference to the production of fat. Catholic Univ. of America Biol. Ser. 13, 1933. LocKwooD, L. B., G. E. Ward, and O. E. May: The physiology of Rhizopus oryzae, Jour. Agr. Research 53: 849-857, 1936. LocKwooD, L. B., and G. E. Ward: Fermentation process for itaconic acid, Ind. Eng. Chem. 37: 405-406, 1945. Mass, J. M.: Personal communication, 1950. Metz, O. : Ueber Wachstum und Farbstoffbildung einiger Pilze unter dem Einfluss von Eisen, Zink und Kupfer, Arch. Mikrobiol. 1: 197-251, 1930. Meyerhof, O.: Energy relationships in glycolysis and phosphorylation, Ann. N.Y. Acad. Sci. 45 : 377-393, 1944. 300 PHYSIOLOGY OF THE FUNGI Meyerhof, O. : Glycolysis of animal tissue extracts compared with the cell-free fermentation of yeast, Wallerstein Labs. Comniuns. 12 : 255-264, 1949. MiCHENER, H. D., and N. Snell: Studies on cultural requirements of Claviceps purpurea and inactivation of ergotamine, Am. Jour. Botany 37: 52-59, 1950. Morton, H. E., W. Kocholaty, R. Junowicz-Kocholaty, and A. Kelner: Toxic- ity and antibiotic activity of kojic acid produced by Aspergillus luteo-virescens, Jour. Bad. 50 : 579-584, 1945. MoYER, A. J., and R. D. Coghill: The laboratory-scale production of itaconic acid by Aspergillus terreus, Arch. Biochem. 7: 1G7-183, 1945. ♦MoYER, A. J., E. J. Umberger, and J. J. Stubbs: Fermentation of concentrated solutions of glucose to gluconic acid. Improved process, Ind. Eng. Chem. 32 : 1379-1383, 1940. Mulder, E. G.: Ueber die Bedeutung des Kupfers fiir das Wachstum von Mikro- organismen und iiber eine mikrobiologische Methode zur Bestimmung des pflanzenverfiigbaren Bodenkupfers, Arch. Mikrobiol. 10 : 72-80, 1939. Myrback, K.: Products of the enzymatic degradation of starch and glycogen. Advances in Carbohydrate Chem. 3, 1948. NoRD, F. F. : Food composition containing Fusaria, U.S. patent 2,450,055, Sept. 28, 1948. NoRD, F. F., and R. P. Mull: Recent progress in the biochemistry of Fusaria, Advances in Enzymol. 5: 165-205, 1945. Owen, W. L. : Production of industrial alcohol from grain by the amylo process, Ind. Eng. Chem. 25: 87-89, 1933. Pasteur, L.: Etudes sur la biere, 1872. Studies on Fermentation. The Diseases of Beer (trans. F. Faulkner and D. C. Robb), Macmillan & Co., Ltd., London, 1879. Payne, E. H., and J. H. Knaudt: Treatment of epidemic typhus with Chloromycetin, Proc. Intern. Cong. Trop. Med. and Malaria, 4th Congr., 4: 426-428, 1948. Peltier, G. L., and R. Borchers: Riboflavin production by molds. Jour. Bad. 64 : 519-520, 1947. Perlman, D., W. W. Dorrell, and M. J. Johnson: Effect of metallic ions on the production of citric acid by Aspergillus niger, Arch. Biochem. 11: 131-143, 1946. Platt, B. S., and R. A. Webb: Fermentation and human nutrition, Biochem. Jour. 39:31, 1945. PoRGEs, N., T. F. Clark, and E. A. Gastrock: Gluconic acid production. Repeated use of submerged Aspergillus niger for semi-continuous production, Ind. Eng. Chem. 32: 107-111, 1940. PoRGEs, N., T. F. Clark, and S. I. Aronovsky: Gluconic acid production. Repeated recovery and re-use of submerged Aspergillus niger by filtration, Ind. Eng. Chem. 33: 1065-1067. 1941. ■^Pratt, R., and J. Dufrenoy: Cytochemical mechanisms of penicillin action. VIII. Involvement of ribonucleic acid derivatives, Jour. Bad. 57: 9-13, 1949. Prescott, S. C, and G. G. Dunn: Industrial Microbiology, 2d ed., McGraw-Hill Book Company, Inc., New York, 1949. Preuss, L. M., E. C. Eichinger, and W. H. Peterson: The chemistry of mold tissue. III. Composition of certain molds with special reference to the lipid content. Cent. Bakt., Abt. II, 89: 370-377, 1934. Raaf, H.: Beitrage zur Kenntnis der Fett- und Eiweissyntheses bei Endomyces vernalis und einigen anderen Mikroorganismen, Arch. Mikrobiol. 12 : 131-182, 1941. Raistrick, H., J. H. Birkinshaw, J. H. V. Charles, P. W. Clutterbuck, F. P. Coyne, A, C. Hetherington, C. H. Lilly, M. L, Rintoul, W. Rintoul, METABOLIC PRODUCTS 301 R. Robinson, J. A. R. Stoyle, C. Thom, and W. Young: Studies in the bio- chemistry of micro-organisms, Trans. Roy. Soc. (London), Ser. B,220: 1-367, 1931. Raistrick, H., R. Robinson, and A. R. Todd: Studies in the biochemistry of micro-organisms. XXXII. Cynodontin (l,4,5,8-tetrahydroxy-2-methylan- thraquinone), a metabolic product of H elminthosporium cynodontis Marigroni and H elminthosporium euchlaenae Zimmermann, Biochem. Jour. 27: 1170-1175, 1933. Raistrick, H., R. Robinson, and D. E. White: Studies in the biochemistry of micro-organisms. L. Ravenelin(3-methyl-l,4,8-trihydroxyxanthone), a new metaboUc product of H elminthosporium ravenellii Curtis and H. turcicum Passerini, Biochem. Jour. 30: 1303-1314, 1936. Rake, G., and R. Donovick: Studies on the nutritional requirements of Strepto- myces griseus for the formation of streptomycin. Jour. Bad. 62 : 223-228, 1946. *Raper, K. B., D. F. Alexander, and R. D. Coghill: Penicillin. II. Natural variation and penicillin production in Penicillium notatum and allied species, Jour. Bad. 48: 639-659, 1944. Raper, K. B., R. D. Coghill, and A. Hollaender: The production and charac- terization of ultraviolet-induced mutants in Aspergillus terreus. II. Cultural and morphological characteristics of the mutations. Am. Jour. Botany 32: 165- 176, 1945. Reilly, H. C, a. Schatz, and S. A. Waksman: Antifungal properties of antibiotic substances. Jour. Bad. 49 : 585-594, 1945. Renaud, J., and M. Lachatltx: Recherches sur la formation de lactoflavine a partir de r Eremothecium ashbyii. Influence des constituants du milieu sur la produc- tion du pigment, Compt. rend. acad. sci. 221 : 187-188, 1945. Rickes, E. L., N. G. Brink, F. R. Koniuszy, T. R. Wood, and K. Folkers: Com- parative data on vitamin B12 from liver and a new source, Streptomyces griseus, Science 108 : 634-635, 1948. RoBBiNS, W. J., A. Hervey, R. W. Davidson, R. Ma, and W. C. Robbins: A survey of some wood-destroying and other fungi for antibacterial activity, Bidl. Torrey Botan. Club 72 : 165-190, 1945. Roberts, M., S. Laufer, E. D. Stew^art, and L. T. Saletan: Saccharification of wheat by fungal amylases for alcohol production, Ind. Eng. Chem. 36 : 811-812, 1944. RoLFE, R. T., and F. W. Rolfe: The Romance of the Fungus World, J. B. Lippin- cott Company, Philadelphia, 1926. Rose, W. C. The nutritive significance of the amino acids, Physiol. Revs. 18: 109-136, 1938. Saito, K.: Ein Beispiel von Milchsaurebildung durch Schimmelpilze, Cent. Bakt., Abt. II, 29: 289-290, 1911. Schatz, A., and E. L. Hazen: The distribution of soil microorganisms antagonistic to fungi pathogenic for man, Mycologia 40 : 461-477, 1948. ScHLEEF, M. L.: The economics of fodder yeast from sulfite waste liquor, Wash. State Coll. Economic and Business Studies Bull. 7, 1948. Schoenbach, E. B., M. S. Bryer, and P. H. Long: The pharmacology and clinical trial of aureomycin: a preliminary report, Ann. N.Y. Acad. Sci. 51: 267-279, 1948. Schopfer, W. H.: Etude et identification d'xm carotinoide de champignon, Compt. rend. soc. biol. 118 : 3-5, 1935. SciARiNi, L. J., R. p. Mall, J. C. Wirth, and F. F. Nord: Concerning the relation between structure and action of xanthones on dehydrogenations by Fusaria, Proc. Natl. Acad. Sci. U.S. 29: 121-126, 1943. 302 PHYSIOLOGY OF THE FUNGI *Shu, p., and M. J. Johnson: Critic acid production by submerged fermentation with Aspergillus niger, Ind. Eng. Chem. 40: 1202-1205, 1948. Smith, E. L. : Purification of antiperinicious anemia factors from liver, Nature 161 : 638-639, 1948. Smith, R. M., D. A. Joslyn, O. M. Gruhzit, I. W. McLean, M. A. PENNER,and J. Ehrlich: Chloromycetin: biological studies. Jour. Bad. 55: 425-448, 1948. Starkey, R. L. : Lipid production by a soil yeast, Jour. Bad. 51 : 33-50, 1946. Sumner, J. B., and G. F. Somers: Chemistry and Methods of Enzymes, 2d ed.. Academic Press, Inc., New York, 1947. *Sure, B. : Nutritional improvement of cereal flours and cereal grains. Jour. Am. Dietet. Assoc. 22: 494-502, 1946. Sure, B. : Further studies on nutritional improvement of cereal flours and cereal grains with yeast, Jour. Am. Dietd. Assoc. 23: 113-119, 1947. Takamine, J.: Enzymes of Aspergillus oryzae and the application of its amyloclastic enzyme to the fermentation industry. Jour. Ind. Eng. Chem. 6: 824-828, 1914. Tanner, F. W., Jr., and M. J. van Lanen: Production of riboflavin by Candida flareri, U.S. patent 2,424,003, July 15, 1947. Chem. Abs. 41 : 6368, 1947. Tanner, F. W., Jr., C. Vojnovich, and J. M. van Lanen: Riboflavin production by Candida species, Science 101: 180-181, 1945. .^Tanner, F. W., Jr., C. Vojnovich, and J. M. van Lanen: Factors affecting ribo- flavin production by Ashbya gossypii, Jour. Bad. 58: 737-745, 1949. Tauber, H.: Chemistry and Technology of Enzymes, John Wiley & Sons, Inc., New York, 1949. Thaysen, a. C: Value of microorganisms in nutrition (food yeast). Nature 151: 406-408, 1943. *Thornberry, H. H., and H. W. Anderson: Synthetic medium for Streptomyces griseus and the production of streptomycin, Arch. Biochem. 16 : 389-397, 1948. Underkofler, L. a., G. M. Severson, and K. J. Goering: Saccharification of grain mashes for alcoholic fermentation, Ind. Eng. Chem. 38 : 980-985, 1946. Utech, N. M., and J. Johnson: The inactivation of plant viruses by substances obtained from bacteria and fungi. Phytopathology 40: 247-265, 1950. Van Lanen, J. M., H. P. Broquist, M. J. Johnson, I. L. Baldwin, and W. H. Peterson: Synthesis of vitamin Bi by yeast, Ind. Eng. Chem. 34: 1244-1247, 1942. Vaughn, J. R., J. L. Lockwood, G. S. Rajstdwa, and G. Hamner: Action of acti- dione on plant tissue and upon certain fungi, Mich. Agr. Expt. Sta. Quart. Bull. 31 : 456-464, 1949. Von Loesecke, H. W. : A review of information on mycological citric acid produc- tion, Chem. Eng. News 23 : 1952-1959, 1945. Von Loesecke, H. W.: Controversial aspects: yeast in human nutrition. Jour. Arji. Dietet. Assoc. 22 : 485-493, 1946. Waksman, S. a.: Associative and antagonistic effects of microorganisms. I. His- torical review of antagonistic relationship. Soil Sci. 43: 51-68, 1937. Waksman, S. A.: Humus, Origin, Chemical Composition and Importance in Nature, 2d ed.. The Williams & Wilkins Company, Baltimore, 1938. Waksman, S. A.: Microbial Antagonism and Antibacterial Substances, Common- wealth Fund, Division of Publication, New York, 1947. Waksman, S. A., and J. W. Foster: Respiration and lactic acid production by a fungus of the genus Rhizopus, Jour. Agr. Research 57 : 873-899, 1939. Walker, T. K. : Pathways of acid formation in Aspergillus niger and in related molds, Advances in Enzymol. 9 : 537-584, 1949. METABOLIC PRODUCTS 303 Wallerstein, J. S. : On the genealogy of beer, Wallerstein Labs. Communs. 2 : 35-45, 1939. *Wallerstein, L. : Enzyme preparations from microorganisms. Commercial pro- duction and industrial application, Ind. Eng. Chevi. 31 : 1218-1224, 1939. Ward, G. E., L. B. Lockwood, O. E. May, and H. T. Herrick: Production of fat from glucose by molds. Cultivation of Penicillium javanicum van Beyma in large scale laboratory apparatus, Ind. Eng. Chem. 27 : 318-322, 1935. Ward, G. E., L. B. Lockwood, 0. E. May, and H. T. Herrick: Studies in the genus Rhizopus. I. The production of dextro-lactic acid, Jour. Am. Chem. Soc. 58: 1286-1288, 1936. Ward, G. E., L. B. Lockwood, B. Tabenkin, and P. A. Wells: Rapid fermentation process for dextrolactic acid, Ind. Eng. Chem. 30: 1233-1235, 1938. Weiss, S., and F. F. Nord: On the mechanism of enzyme action. XXXVIL Solanione, a pigment from Fusarium solani D2 purple, Arch. Biochem. 22 : 288-313, 1949. Wells, P. A., A. J. Moyer, and O. E. May: The chemistry of citric acid fermenta- tion. I. The carbon balance. Jour. Am. Chem. Soc. 58: 555-558, 1936. Wells, P. A., A. J. Moyer, J. J. Sti^tbbs, H. T. Herrick, and O. E. May: Gluconic acid production. Effect of pressure, air flow, and agitation on gluconic acid production by submerged mold growths, Ind. Eng. Chem. 29: 653-656, 1937. West, R. : Activity of vitamin B12 in Addisonian pernicious anemia, Science 107: 398, 1948. Whiffin, a. J.: The activity in vitro of cycloheximide (acti-dione) against fungi pathogenic to plants, Mycologia 42 : 253-258, 1950. WiELAND, H., and B. Witkop: Giftstoffe des Knollenblatterpilzes. V. Zur Kon- stitution des Phalloidins, /. Liebigs Ann. d. Chem. 543: 171-183, 1940. WiLKiNS, W. H., and G. C. M. Harris: Investigations into the production of bac- teriostatic substances by fungi. VI. Examination of the larger Basidiomycetes, Ann. Applied Biol. 31 : 261-270, 1944. WoHLER, F.: Das Entrathselte Geheimniss der geistigen Garung (Vorljiufige brief- Hche Mittheilung), J. Leibigs Ann. d. Chem. 29 : 100-104, 1839. Wolf, F. A., and F. T. Wolf: The Fungi, Vol. II, John Wiley & Sons, Inc., New York, 1947. Wright, L. T., M. Sanders, M. A. Logan, A. Prigot, and L. M. Hill: The treat- ment of lymphogranuloma venereum and granuloma inguinale in humans with aureomycin, Ann. N.Y. Acad. Sci. 151: 318-330, 1948. Ytjill, J. L. : Alcoholic fermentation by Aspergillus flavus Brefeld, Biochem. Jour. 22: 1504-1507, 1928. CHAPTER 14 FACTORS INFLUENCING SPORULATION OF FUNGI The life of the individual fungus is usually short and of uncertain dura- tion. The continuance of the species (in most instances) depends upon the production and dissemination of sexual or asexual spores. The importance of spore production in the spread of epiphytotics is sufficient reason to study the factors which control, modify, or inhibit this stage of development in the life of the fungi. Some of the most difficult problems which arise in the study of the life processes of the fungi are to be found in the events and conditions which control the production of spores. In nature, we find many examples of the influence of certain environ- mental and nutritional factors upon reproduction of the fungi. A num- ber of parasitic fungi produce the perfect stage only in the spring, on or in dead host tissue. This is true of Venturia inaequalis, Clavtceps purpurea, Gnomonia ulmi, Monilinia jructicola, Coccomyces hiemalis, Guignardia bidwelln, and others. Is the production of the sexual stage dependent upon the pretreatment of cold, or freezing and thawing? Is it dependent upon a favorable temperature and perhaps favorable intensity and dura- tion of light? Or is it a matter of the proper nutrients which are made available only after decay of the host tissues? These are difficult ques- tions to answer, for it is likely that the production of the sexual stage depends upon the proper balance of a number of factors. Similarly, we may speculate about the stimuli involved in the formation of the peri- thecia by the Erysiphales. Most of these obligate parasites form fruit bodies late in the growing season. Perhaps, at least in some cases, this is a reaction to cooler weather ; or perhaps the formation of perithecia is a result of a decreasing or changing food supply as the host nears maturity. Other physical factors are probably involved, since we know that the abundance of perithecia varies from year to year. It is also of physio- logical interest that many parasitic fungi produce conidia only while the mycelium is actively attacking the living host. A critical investigation of the factors influencing reproduction requires that the fungi be brought into the laboratory or greenhouse where external factors can be controlled. Only one variable should be studied at a time, and all other influencing factors must be controlled. It is, therefore, of great advantage in physiological studies to be able to grow a fungus in pure culture on synthetic or semisynthetic media. However, it n\ust be 304 SPORULATION 305 pointed out that the responses of a fungus in nature cannot always be duplicated in the laboratory. Snyder and Hansen (1947) have given a brief and clear statement regarding the advantages of culturing fungi on natural media and under natural environmental conditions. These conditions are important, if one desires to obtain reproduction of a fungvis which does not sporulate readily in culture. However, if one desires to study critically the indi- vidual nutritional and environmental requirements and their effects upon reproduction of a fungus which sporulates abundantly on the usual cultural media, it is often necessary to subject the fungus to unfavorable conditions. Thus, only by preventing sporulation, by varying but one factor at a time, may we discover the need for that factor. Riker and Riker (193G) have listed 11 methods which have been suc- cessfully employed to induce sporulation of different fungi in culture. Since the writing of their manual much has been learned about this phase of fungus physiology. A revised list of the conditions known to influence sporulation of fungi is presented in the summary of this chapter. Kauffman (1929) called attention to the views of Klebs, who held that living cells are influenced during their lifetime in three ways: (1) by the specific structure; (2) by the internal conditions; and (3) by the external conditions. Kauffman equated the first of these to heredity and the last two to environment. The external environment comprises the various physical and chemical factors, such as temperature, light, composition of the medium, and the like. Kauffman used the term internal environ- ment to designate the complicated influences and reactions between cells wdthin the organism. The physical and chemical effects of the external environment may be transmitted through the cells and become evident at some distance from the point of the stimulus. The meaning of these statements may be clearer if we consider the effect of various external environmental factors upon fruiting. It is well known that various external stimuli may initiate the reactions which lead to reproduction. These stimuli must act through the internal environ- ment. Most of the discussion that follows will be concerned with the external environment and the resulting development of the fvmgus. Some external factors may so modify the internal milieu as to favor sporulation, w^hile others may inhibit or prevent sporulation. Not all fungi respond in the same way to the external factors such as light, temperature, or nutrition. Each species produces spores when the internal environment is suitable, but the external factors do not operate upon the internal environment of all fungi alike. Thus, there is no univer- sal set of external conditions which lead to fructification in all fungi. The external conditions favorable for sporulation must be studied for each species. This does not imply, however, that no two fungi react alike or 306 PHYSIOLOGY OF THE FUNGI that certain helpful generalizations concerning sporulation cannot be drawn. It does, however, imply that the only sure way of understanding the conditions governing reproduction in a specific fungus lies in the experimental approach. Again it must be emphasized that all the physical and chemical condi- tions may be at the optima, but no reproduction can occur without the presence of favorable genetic factors. Too often we may fail to realize the genetic requirements. The appropriate steps should be taken to determine whether the fungi under study are homothallic or heterothallic. It may be difficult, indeed, to determine whether failure of a fungus to reproduce in culture is due to unfavorable environmental conditions or to unfavorable genetic factors. There is much yet to be learned regard- ing the physiology of reproduction, but each new investigation is certain to add to our knowledge of this interesting and important phase of fungus physiology. Vegetative growth must precede reproduction. The length of the vegetative phase varies from organism to organism, and the same organ- ism may remain in the vegetative phase for a longer or shorter period of time depending upon the external environment. One of the functions of the vegetative phase is concerned with the building up of protoplasm and the storage of energy reserves. Reproduction is a process that draws heavily on the reserve food. The spore is usually well stocked with these materials. Asexual reproduction differs less from vegetative growth than does sexual reproduction. We shall find that the conditions limiting- sexual reproduction are usually more narrow than conditions which allow asexual reproduction and growth. Klebs (1900) summarized his views on reproduction in the fungi in the form of four laws or principles as follows: (1) Growth and reproduction are life processes, which, in all organisms, depend upon different condi- tions. In the lower organisms the external conditions mainly determine whether growth or reproduction takes place. (2) Reproduction in the lower organisms does not occur as long as characteristic external condi- tions are favorable for growth. The conditions which are favorable for reproduction are always more or less unfavorable for growth. (3) The processes of growth and reproduction differ, in that growth may take place under a wider range of environmental conditions than reproduction. Growth may take place, therefore, under conditions which inhibit repro- duction. (4) Vegetative growth appears to be mostly a preliminary step for reproduction in that it creates a suitable internal environment for it. To a certain degree it is not growth in itself but the prolonged period of assimilation accompanying growth that is decisive for reproduction. These generalizations were published in 1900 and were based upon Klebs's own work, as well as that of others. Many more fungi have been SPORULATION 307 studied during the past 50 years, and some new factors have been brought to light. It would not be surprising if some modifications in these con- clusions would be necessaiy in the light of 50 years of research. We shall find, however, that, in the main, many of these "laws" are still valid. ENVIRONMENTAL FACTORS Temperature. Temperature was recognized by Bisby (1943) as an important natural factor governing the geographical distribution of the fungi. The temperature must be favorable not only for growth but also for the production and germination of the spores, if the fungus is to survive. Certain fungi are limited by high temperatures. Among these are Plasmodiophora brassicae, Colletotrichum lindemuthianum, Urocystis cepulae, and certain Phycomycetes. On the other hand, certain genera of the Gasteromycetes, such as Podaxis, Battarrea, Chlamydopus, and Phellorina, are confined to the hot arid regions of southwestern United States, northern Africa, central Australia, and western India. Between these extremes we may observe many examples where seasonal tempera- ture limits or favors reproduction. Klebs (1900) pointed out that the temperature range which allowed sporulation was more narrow than the range for growth. In general, the temperature limits for sexual reproduction are narrower than the limits for asexual reproduction. Some of Klebs's data are presented in Table 53. Table 53. Minimum and Maximum Temperatures (in Degrees Centigrade) for Growth axd Sporulation of Various Fungi (Klebs, Jahrb. iriss. Botan. 35, 1900.) Fungus Aspergillus repens. . . Sporidinia grandis. . . Piloholus rnicrosporus Saprolegnia mixta. . . . Growth Min. 7-8 1-2 2-4 0-1 Max. 37-38 31-32 33-34 36-37 Asexual spores Min. 8-9 5-6? 10-12 1-2 Max. 35-36 29-30 28-30 32-33 Sexual spores Min. 5-6 1-2 Max. 33-34 27-28 26-27 It will be noted that the upper temperature which allowed the produc- tion of oospores by Saprolegnia mixta is a full 10°C. less than the upper temperature limit at which growth took place. Coons (1916) found the temperature limits for the growth of Plenodomus fuscomacidans to be 0 to 33°C., while pycnidia formed between 6 and 30°C. Perithecia failed to form in cultures of Ceratostomella fimbriata kept at 18°C. for 60 days (Barnett and Lilly, 1947a). Cultures of this fungus on the same medium 308 PHYSIOLOGY OF THE FUNGI produced abundant perithecia and ascospores at 25°C. within 11 days. Conidia were formed at 18°C. The most noteworthy effect of culturing a fungus at temperatures below the optimum is the decrease in the rate of growth. It has been found by various investigators that there is an optimum temperature for sporulation as well as for growth. The two optima may be different. Figure 57 shows the effect of temperature on the time required to produce conidia by Aspergillus repens. 14 12 10 2 6 Q, 4 o o • \ \ L \ \ •\ V \ • i 1 X. — .- y 10 15 20 25 30 Temperature in degrees centigrode 35 40 Fig. 57. The influence of temperature on the time required to produce conidia by Aspergillus repens. (Drawn from data of Klebs, Jarhh. wiss. Botan. 35 : 137, 1900.) A temperature of 28°C. was optimum for sporulation of Piricularia oryzae (Henry and Andersen, 1948). Higher and lower temperatures of incubation decreased the numbers of spores produced. At 32°C. the number of spores was only 10 to 15 per cent of that produced at the optimum temperature. Reducing the temperature of incubation to 24°C. reduced the numbers of spores to about 80 per cent of the maxi- mum. Thus, a small temperature increase above the optimum has a much greater effect upon the number of spores produced than a small decrease in temperature below the optimum (Fig. 58). In nature, fungi are exposed to fluctuating temperatures. Whether a fluctuating temperature is more favorable in inducing sporulation than a constant temperature appears to have been studied but little. Jones (1946) concluded that temperature was the important controlling factor in the production of resistant sporangia of Allomyces arhiiscula in culture, and he beheved that "the total amount of temperature" to which the cultures were subjected was more important than the maximum, mini- SPORULATWN 309 mum, or degrees of fluctuation, IMathur et al. (1950) reported that 15 to 20°C. favors conidium formation by Colletotrichum lindemuthianum in culture. Sporulation was less at 25°C. and ceased at 30°C. Mrak and Bonar (1938) found that temperature influenced the relative size of asci and spores of Debaryomyces. The ascus was much larger than the spore cluster at 4°C., but the spores nearly filled the ascus at 25°C. 13 15 7 9 11 Incubation period (days) Fig. 58. The effects of temperature and time of incubation on sporulation of Piri- rularia oryzae on rice-polish agar. (Courtesy of Henry and Andersen, Phytopathology 38 : 272, 1948.) An interesting selective effect of temperature upon type of asexual sporulation is found in Choanephora cucurbitarum (Barnett and Lilly, 1950). This fungus produces two types of asexual spores, those produced in typical sporangia and conidia borne in heads. Only the conidia are found commonly in nature, while both types are abundant in culture. When the fungus was grown in Petri dishes at 25°C., 87 per cent of the reproductive structures were conidial heads, while 13 per cent were sporangia (Table 54). When the temperature was increased to 30°C., this proportion was nearly reversed. At 31°C. many sporangia but no conidia were formed. No sporulation occurred at 34°C., but mycelial growth was abundant. Temperature also affected, either directly or indirectly, the size of the sporangia. Those produced at 25°C. averaged 60 to 90 n in diameter, while those formed at 30 or 31°C. were much larger, averaging approximately 145 fi. It seems likely that this effect is indirect, being a reflection of the relative number of conidia, which are formed first under favorable conditions. We may assume that the production of abundant conidia uses much of the food materials which might also go 310 PHYSIOLOGY OF THE FUNGI into the formation of sporangia. Under conditions unfavorable to conidium production, yet favorable to sporangium formation, both the size and abundance of sporangia are increased. The effect of temperature was also evident when pumpkin flowers artificially inoculated with C. cucurbitarum were brought into the laboratory and placed at 30°C. Under these conditions both conidia and sporangia were produced. Table 54. The Effect of Temperature upon Asexual Reproduction op Choanephora cucurbitarum (Barnett and Lilly, Phytopathology 40: 83, 1950.) Temperature during sporulation, °C. Conidial heads per culture Sporangia per culture Average size of sporangia, fx 25 30 31 34 2,000 150 0 0 300 1,300 1,200 0 60-90 148 145 Other critical temperature studies are needed, particularly those designed to show the interrelated effects of temperature with other environmental or nutritional factors and to determine the effects of tem- perature upon the "internal environment" of the fungi. The tempera- ture of incubation affects zygospore formation by Phycomyces blakes- leeanus indirectly through the amount of acid formed in the medium (Robbins and Schmitt, 1945). Light. Light has been a neglected and often ignored factor in many studies of sporulation. Too often we place fungi in the laboratory or refrigerator according to our own convenience, not to their needs, and expect them to reproduce as they would in nature. Under natural con- ditions many fungi fruit only when exposed to light, often to the direct rays of the sun, for a part of the time. Numerous observations have been reported regarding the need for light, but too few of these reports give data as to the intensity, duration, or quality of the light required to initiate sporulation. We should not conclude that intensity and duration are without effect. A review of the early work on the influence of light on the growth and fruiting of the fungi is presented by Coons (1916). Brefeld (1877) found that some species of Coprinus failed to fruit in the dark. A culture of Coprimis exposed to light for 2 or 3 hr. was then able to fruit in the normal manner when removed from the light. He also found that higher tem- peratures replaced, in part, the beneficial effect of light for some species. Sphaerographium fraxini produced a few pycnidia in the dark at 30°C., whereas none were produced at room temperature in the dark (Leonian, 1924). Pycnidia were produced at room temperature in the light. SPORULATION 311 Ascochyta nymphaeae, Cytosporella mendax, Endothia parasitica, Keller- mania yuccagena, Naemosphaera sp., Plenodomus destruens, and Phoma urens formed more pycnidia at 30°C. in the dark than in the light at room temperature. The following fungi failed to fruit in the dark at 8°C. but fruited at the same temperature in the presence of light: Hendersonia sp., Melanconium hetulinum, Naemosphaera sp., Pestalotia guepinia, Phoma urens, Phyllosticta opuntiae, Sphaerographium fraxini , and Sphaero- nema pruinosum. Light favored pycnidial formation by Plenodomus fuscomacidans (Coons, 191G). The above examples make it clear that light and temperature may serve as interchangeable stimuli to sporulation in some, but not all, instances. Since the response (sporulation) is the same whether light or temperature is the stimulus, this means that these stimuli in some way brought about the same or equivalent changes in the internal environment of the fungus. Drayton (1937) was able to produce the perfect stage of Botryotinia convoluta by controlling light, temperature, and nutrition. The technique is somewhat involved, but it should be remembered that in nature the external environment varies a great deal during the course of a year. Fluctuations in temperature, moisture, light, and food supply are the normal result of the procession of the seasons. Drayton found autoclaved W'hole wheat to be an excellent substratum for this fungus. The most favorable results w^ere obtained by allowing the culture to develop at 14°C. in the dark for 45 days. At the end of this time the sclerotia w^ere placed in moist quartz sand at 0°C. for 3 to 4 months, then stored at 5°C. When the apothecial fundaments were 2 to 3 mm. long, the cultures were moved to a greenhouse and placed under cheesecloth and the tem- perature held at 7°C. at night and below 15°C. during the day. The apothecia matured within 4 wrecks. Yarwood (1936, 1941) observed parasitic fungi under natural condi- tions and found that the production and liberation of the conidia of Erysiphe polygoni and the ascospores of Taphrina deformans followed a definite diurnal pattern in nature. The combined effects of temperature and light upon sporulation of Helminthosporium gramineum are clearly shown by Houston and Oswald (1946). Best sporulation was obtained under outdoor conditions, with 14 to 15 hr. of daylight and the average maximum and minimum tem- peratures 26.8 and 8.2°C., respectively. No conidia were produced on potato-glucose agar in the absence of light, either outdoors or inside. Artificial light apparently was less effective than daylight. However, continuous light at 13°C. allowed the formation of a few conidia. On pieces of infected barley leaves, conidia were formed without exposure to light, over a considerable range in temperature. As an explanation of these differences, the authors believe that the mycelium in the leaf in 312 PHYSIOLOGY OF THE FUNGI nature stored up the "necessary potentialities," which then permitted conidium production in darkness. MyceHum growing from the pieces of leaf into the agar did not produce spores in darkness. This is an interesting theory regarding a possible delayed action of light upon sporu- lation. It also seems possible that the leaf tissue of the host may furnish some nutrient necessary for sporulation which is not contained in potato- glucose agar. Perhaps light is essential to the synthesis of this material by the fungus. It was demonstrated recently (Barnett and Lilly, 1950) that an isolate of Choanephora cucurhitarum requires both light and darkness for the forma- tion of conidia, but these factors have little or no apparent influence upon the formation of sporangia. This fungus was grown under a number of conditions, but none was found which overcame the need for either light or darkness. Cultures incubated in the laboratory under natural alter- nating light and darkness produced abundant conidial heads during the second and third nights after inoculation. Exposure to artificial light for 2 days after inoculation followed by darkness gave similar results, but an exposure in the reverse order resulted in no conidia. Cultures under continuous artificial light (65 foot-candles) and those in total continuous darkness failed to form conidial heads. Continuous light of low intensity (less than 1 foot-candle) , however, did permit the formation of numerous conidial heads in the usual period. A summary of the important results is presented in Fig. 59, together with an outline of a proposed hypothesis to explain the results. We may assume that light, or its absence, affects two metabolic reactions, or groups of reactions, which are essential to conidium formation by C. cucurhitarum. Light, which is essential to reaction A, apparently inhibits reaction B, which must occur in darkness or weak light. The reaction in light must be'^followed by the reaction in darkness, if conidia are to be formed. Continuous bright light favors only reaction A, while continuous darkness permits only reaction B. Both reactions occur simultaneously in continuous light of low intensity. A different isolate of C. cucurhitarum was studied by Christenberry (1938), who found that alternating periods of light and dark, 12 hr. each, gave the best sporulation. Red-yellow light was more favorable to conidium formation than the shorter rays. This isolate formed conidia in total darkness. The beneficial effect of alternating light, or a period of light followed by darkness, was demonstrated (Timnick et al., 1951) for the formation of ascospores by Diaporthe phaseolorum var. hatatafis. Cultures grown in continuous darkness formed only a few perithecia, which contained abundant ascospores. In continuous bright light numerous perithecia were formed, but relatively fewer ascospores were produced. A long period of light followed by darkness gave many perithecia with abundant ascospores. SPORULATION 313 Marked morphologic differences were found in strains of Fusarium subjected to different exposures of light and darkness (Snyder and Han- sen, 1941). Some of the characters affected were color, zonation, type of colony, presence or absence of sporodochia, occurrence of the perithecial stage, and size, shape, and septation of macroconidia. Light was usually found necessary for the formation of macroconidia. Exposures were made to continuous total darkness but not to continuous light. Evidence in these experiments indicated that the effect of light is only upon the actively growing portion of the mycelium. Bright light Reaction A Darkness Reaction B = Conidia Continuous bright light Reaction A Continuous darkness Reaction B = No conidia = No conidia Bright light Reaction A = No conidia Continuous light low intensity Conidia Reactions A + B simultaneously Fig. 59. Conidium formation by Choanephora cucurbitarum under different light conditions, shownig the possible metabolic reactions controlled by light. Under variable conditions, the cultures were exposed to the first condition (on the left) for 2 days and to the second condition for 24 hr. (After Barnett and Lilly, Phytopathology 40: 88, 1950.) The length of exposure necessary to stimulate spore formation may be very short, as demonstrated by Bisby (1925) for Fusarium discolor sul- phureum. He observed that brief exposure to light, while Petri dish cultures were being examined, resulted in the formation of rings of conidia. Using a photographic shutter, he further demonstrated that an exposure as brief as }i sec. to outdoor light on a bright day was sufficient to stimu- late the formation of a ring of conidia. Coons (1916), in his work with Plenodomus fuscomacidans, reasoned that the effect of light might be replaced by various oxidizing agents, since light is known to promote various oxidations. Cultures treated with hydrogen peroxide and other oxidizing agents produced a few pycnidia. The age of the culture when these chemicals were added was important. 314 PHYSIOLOGY OF THE FUNGI For these chemicals to stimulate pycnidium formation, the culture had to be in such a physiological condition that 1-hr. exposure to light would induce sporulation. The sporulation of a number of other species in our laboratory has been observed to be influenced by the presence or absence of light (Figs. 60, 61). Among these are Dendrophoma obscurans, Trichoderma lignorum, Fig. 60. The effects of light on the production of conidia by Trichoderma lignorum after 3 days at 25°C. A, exposed to continuous artificial light. Note the more or less even distribution of conidia. B, exposed to alternate ight and darkness, 12 hr. each. Note the rings of conidia. C, grown in continuous darkness. Note the ab- sence of conidia. Sphaeropsis malorum, Ceratostomella ulmi, Botrytis sp., Endothia para- sitica, Septoria nodorum. The reaction of some fungi to light is appar- ently dependent, to a certain extent, upon the composition of the medium. Still another effect of light should be emphasized, i.e., the inhibitory effect. The depressing effect of strong light upon growth and length of sporangiophores of Phy corny ces hlakesleeanus is easily demonstrated. Elfving (1890) noted that the amount of inhibition of growth by light varied with the composition of the medium. Ultraviolet light. The destructive action of sunlight upon micro- organisms, especially bacteria, was recognized about the time that pure- SPORULATION 315 culture methods came into wide use. The lethal action of ultraviolet light is conditioned by the wave length of the irradiation, by the time of exposure, and by the particular nature of the microorganism. A con- siderable number of investigators have studied the effect of ultraviolet radiation upon sporulation. Both favorable and unfavorable results have been obtained. It should be recognized that length of exposure Fig. 61. The effect of light on the production of pycnidia by an isolate of Dendro- phoma obscurans when grown on malt extract-agar plates at 25°C. A, grown under continuous artificial light, .\lternate light and darkness gave similar results. B, grown in continuous darkness. is a very important factor in these experiments. In addition, the medium used, the age of the culture, and the temperature rise during irradiation also modify the results. Stevens (1928) found that ultraviolet radiation induced the formation of perithecia by various isolates of Glomerella cingulata a few days after irradiation. While old cultures produced a few perithecia without irradiation, many more were produced by young cultures within a short time following irradiation. One effect of such irradiation is the killing of the aerial mycelium. Short exposures allowed the formation of super- ficial perithecia, while long exposures prevented their formation. The majority of the perithecia formed following intermediate dosages were embedded in the medium. It was noted that the age of the mycelium 316 PHYSIOLOGY OF THE FUNGI at the time of irradiation had an effect on the number of perithecia formed. Colonies 4 days old when irradiated produced perithecia, which were most abundant on mycelium 1 day old at the time of irradiation. Irradiation of colonies 12 days old led to the formation of but few perithecia. No evidence was obtained that irradiation of the medium alone had any effect on perithecium formation. A species of Coniothyrium which formed pj^cnidia only after the cultures were very old was stimulated to produce pycnidia within 3 days after irradiation. This work of Stevens is apparently the first which demonstrated that ultraviolet radiation stimulated sporulation by fungi. Spore production by Macrosporium tomato and Fusarium cepae was greatly increased by the proper exposure to ultraviolet radiation (Ramsey and Bailey, 1930). A 12- to 15-fold increase in the numbers of spores produced by these two species was obtained by the optimum exposure. These investigators also showed that irradiation of the medium before inoculation had no subsequent effect on sporulation by these two fungi. The range of wave lengths which stimulated the most abundant sporula- tion was found to be 2,300 to 2,800 A. Smith (1935) points out that many workers have neglected the precaution of controlling the tempera- ture of cultures during irradiation. She found it necessary to control the temperature of the cultures of Fusarium eumartii in order to separate the effects of increased temperature and ultraviolet radiation. Ultraviolet radiation stimulated or depressed sporulation of Diaporthe phaseolorum var. hatatatis depending on the medium used (Timnick et al., 1951). Neither stromata nor perithecia were formed on casein hydrolysate-glucose medium, unless the cultures were irradiated. Cul- tures grown on potato-glucose agar produced stromata and long-beaked perithecia without irradiation. Irradiation of cultures on potato-glucose medium resulted in the formation of fewer and smaller short-beaked perithecia. Although the mode of action of ultraviolet radiation in stimulating sporulation is unknown, long exposures are known to be lethal. We may assume that even short exposures injure or kill some of the exposed cells. Perhaps some substance is thereby released which stimulates sporulation. The presence of such a substance in the potato- glucose medium might explain why irradiation was not necessary for the production of perithecia by D. phaseolorum var. hatatatis on this medium Aeration. Although the fungi are aerobic organisms, the amount of free oxygen that they need to carry out their life processes varies from fungus to fungus. The amount of oxygen required is less for growth than for reproduction. The aquatic fungi would be expected to grow and reproduce in a more limited supply of oxygen than terrestial forms. While many aquatic Phycomycetes produce their spores under water, a large number of fungi fail to fruit until some aerial mycelium has been SPORULATION 317 formed. Examples of the inhibiting effect of insufficient aeration on sporulation are numerous. Coons (1916) found that lowered oxygen ten- sion inhibited pycnidium formation by Plenodomusfuscomaculans, though there was still sufficient oxygen supply to allow some growth. Leonian (1924) tested the effect of reduced oxygen on pj^cnidium formation by various Sphaeropsidales. This experiment was carried out by culturing these fungi in Petri dishes, some of which were placed in desiccators, while the controls were placed on a table. The following fungi produced fewer pycnidia in sealed desiccators than in the control cultures: Ascochyta tiym'phaeae, Phoma urens, Plenodomus destruens, Phyllosticta opuntiae, and Septosporium acerinum. It is possible that this effect may have been due to the increased concentration of carbon dioxide in the closed vessels. Denny (1933) made an accurate study of the effect of oxygen supply on growth and formation of perithecia by Neurospora sitophila. Only a trace of oxygen was required for limited growth, for it was necessary to keep cultures in the presence of alkaline pyrogallol to inhibit growth entirely. Oxygen concentrations of less than 0.5 per cent inhibited perithecium formation for 30 days, while perithecia formed in air within 4 days. This paper should be consulted for the details of conducting experiments of this nature under closely controlled conditions. Some of Denny's data are given in Table 55. Table 55. The Effect of Oxygen Concentration on the Formation of Peri- thecia BY Neurospora sitophila (Prepared from the data of Denny, 1933. Contribs. Boyce Thompson Inst. 5, 1933.) Oxygen Cbncentration, % Days Required to Form Perithecia 20.8 4 9.4 7 3.75 9 1.5 12 0.24 None at 30 days Conidium production by Choanephora cucurhitarum was poor in tight- fitting Petri dishes (Barnett and Lilly, 1950). Sealing the dishes pre- vented conidium formation, while well-aerated dishes allowed abundant conidial heads to form. Failure to form conidia under these conditions may be due to (1) insufficient oxygen supply, (2) the accumulation of toxic, volatile, metabolic by-products, (3) increased carbon dioxide con- tent, or (4) unfavorable humidity. Adequate aeration was one of the most important environmental fac- tors necessary for conidium formation by Piricularia oryzae (Heniy and Andersen, 1948). The cultures emitted a strong odor of ammonia after a few days' incubation. It was believed that aeration removed the ammonia and other volatile metabolic by-products which prevented 318 PHYSIOLOGY OF THE FUNGI abundant sporulation. Forced aeration of the culture flasks at the rate of 4 ml. of air per minute per milligram of oats-sorghum medium was found to be optimum for sporulation. Mader (1943) discussed the factors inhibiting fruiting of Agnricus campestris and concluded that volatile substances are important, and that they must be removed by aeration of mushroom cellars. Hydrogen-ion concentration. The early workers recognized that the acidity of the medium influenced sporulation. Lock wood (1937) studied the formation of perithecia and asci by Penicillium javanicum, Aspergillus herhariorum, and Chaetomium. globosum in buffered media of various Fig. 62. The effect of glutamic acid on gametic reproduction of Phycomyces hlakes- leeanus at 26°C. Left, basal medium; right, basal medium plus 10 mg. d-glutamic acid, neutraHzed with CaCOs. Note the line of progametes in the plate on the right. Age, 6 days. (Courtesy of Robbins and Schmitt, A7n. Jour. Botany 32 : 321, 1945.) hydrogen-ion concentrations and found that the perithecia produced in the more acid solutions contained few if any asci with ascospores. The percentage of fertile perithecia increased as the pH was increased to 7 or 8. Similarly, in our laboratory, we have noted that A . rugulosus produces many perithecia and few conidia at an initial pH value of 6 to 8, while conidia but no perithecia form at pH 3 to 4. Robbins and Schmitt (1945) studied the sexual reproduction of Phyco- myces hlakesleeanus on glucose-asparagine medium and found that mature zygospores did not form at 26°C. Zygospores formed when various protein hydrolysates, amino acids (especially glutamic acid), or various organic acids were added to the medium. These buffers prevented the pH from falling low enough to inhibit zygospore formation (Fig. 62). These authors also noted that P. hlakesleeanus on glucose-asparagine medium produced zygospores at 20°C. This is evidence that the com- position of the medium has a profound effect on reproduction. In this SPORULATION 319 instance, it was possible to trace the connection between temperature and the composition of the medium to a specific factor, i.e., acidity Perithecia were not formed by Sordaria fimicola until the pH of the culture medium was 6.5 or greater (Lilly and Barnett, 1947). While acidity of the medium was not the only controlling factor affecting the formation of perithecia by S. fimicola, perithecia never formed when the pH was less than 6.5, however favorable the other external conditions were. OTHER PHYSICAL FACTORS It has frequently been observed that many species of fungi fruit more readily when grown upon a solid or semisolid substratum than they do in liquid media. Leonian (1924) reported that only 6 out of 20 species studied formed pycnidia as readily in liquid medium as on solid medium. He concluded that the beneficial effect of solid media was due to better aeration and free transpiration. The favorable effect of ozone upon the formation of pycnidia and spores of a limited number of fungi was recently reported by Richards (1949). The production of viable conidia of three species of Alternaria was greatly increased on exposure to ozone. Although conidium formation of Mycosphaerella citrullina was increased by exposure to ozone, the spores formed did not germinate. The transformation and elongation of basidia of certain Polyporaceae in nature and under controlled conditions has been correlated with high humidity by Bose (1943). It seems likely that the humidity of the atmosphere may have a greater influence upon conidium formation in the aerial fungi than is generally supposed. In Rhizopus, for instance, much more liquid moves upward through the sporangiophore than can be con- tained within the sporangium. A high percentage of this water must be transpired in order to condense the protoplasm and food materials stored in the spores. A change in relative humidity must affect the rate of transpiration. On the other hand, Ternetz (1900) found that a humidity of 98 per cent or higher was necessary for fruit-body production by Ascophanus carneus. Actually, we know little about the influence of humidity, and much more information is needed on this subject. Emerson and Cantino (1948) showed that the presence of high concen- trations of carbon dioxide favored the production of resistant sporangia by Blastocladia pringsheimii. Mutilation of the mycelium, which would cause the death and release of cellular constituents, has been used to stimulate sporulation (see Rands, 1917; Kunkel, 1918; and McCallan and Chan, 1944). Scraping of the mycelium of Alternaria solani followed by a brief exposure to ultraviolet rays was used successfully by McCallan and Chan (Fig. 63). 320 PHYSIOLOGY OF THE FUNGI 10,000 (1 '^\ [1 / L [\ { v[ o ^ V. ~^^^ •z ^ -^ — ' 50 Asper grown gillus n on cellc ger Dphane dipped 1 n malt 70 95 100 75 80 85 90 Relative humidity in percent Fig. 74. Germination curves for Aspergillus niger under variable temperature and humidity. Note that the optimum temperature for germination varied with the relative humidity, being near 30°C. at relative humidity of 100 per cent and near 40°C. at 93 per cent. As the temperature or humidity digressed from the optimum, ^the time required for germination increased. (Courtesy of Bonner, Mycologia 40 : 733, 1948.) of about 24 per cent in volume of the Erijsiphe conidia during germina- tion. Spores of all other fungi (except other powdery mildews) which he tested showed increases in volume during germination. Dormancy of some spores may be broken by alternate wetting and drying. This treatment apparently makes the thick resistant wall more permeable to water. Oxygen supply. Since respiration is greatly accelerated during spore germination, it follows thg^t an adequate supply of oxygen is a prerequisite 360 PHYSIOLOGY OF THE FUNGI for germination. Brief reports of a number of observers on oxygen requirements are given by Doran (1922). It is generally agreed that reduced oxygen supply decreases spore germination. Spores germinate better on or near the surface of a li(iuid than when submerged deep in the liquid. In some cases the spores may germinate under water, but only abnormal germ tubes are formed. Aerated water gives better germina- tion than nonaerated water. The spore load in a drop of water, whether all of the same species or of mixed spores, influences greatly the percent- age of germination. This is believed to be due primarily to the competi- tion for the limited supply of oxygen, rather than to toxic substances produced by other germinating spores. According to Jones (1923), spore germination of Ustilago avenae is greatest in soil with 30 per cent of water-holding capacity and is greatly reduced at 80 per cent. This was probably due to the amount of avail- able oxygen. The spores failed to germinate in water when exposed to an oxygen-free atmosphere. The " chlamydospores" of Ustilago zeae do not germinate in the absence of oxygen, and at least 5 per cent oxygen must be present to allow germination as high as in the open air (Platz et at., 1927). The supply of oxygen may influence the method of spore germination (Uppal, 1926). Germination by zoospores was possible in the absence of oxygen for the sporangia of Phytophthora mfcstans, P. colocasiae, P. palmivora, and P. parasitica. Germination by germ tubes does not take place in these species in the absence of oxygen. However, the presence of oxygen is essential for zoospore formation by sporangia of Alhiigo Candida, Plasmopara viticola, and Sclerospora graminicola. The two methods of germination are different processes, the direct method more nearly resembling vegetative growth. Hydrogen-ion concentration. Under natural conditions acidity is not usually a limiting factor for spore germination. In general, spores will germinate within a wide pH range. It seems significant that, in most species of fungi, germination is favored by an acid medium, often at a pH considerably lower than the optimum for vegetative growth or sporu- lation. The effects of acidity of the medium upon a number of species, including Botrytis cinerea, Aspergillus niger, Penicillium cyclopium, P. italicum, Puccinia graminis urediospores, Lenzites saepiaria, Colleto- trichum gossypii, and Fusarium sp., are reported by Webb (1921). The spores of the Fusarium germinated equally well in alkaline and acid media, while CoUetotrichuni gossypii was the only species of the group studied in which germination was better in an alkaline medium. At pH 2.5 spore germination was prevented in all species, and the optimum for most species was 3.0 to 4.0. In sucrose-nitrate (Czapek's) solution, two maxima usually occurred, the primary one at pH 3.0 to 4.0 and a SPORE GERMINATION 361 secondary one between G.O and 7.0. Of all the media tested, beet decoc- tion gave the maximum germination imder the widest range of conditions. Webb also clearly demonstrated that the range of pH favoring germina- tion is influenced by temperature and by the constituents of the medium. All the species of Myxomycetes studied by Smart (1937) germinated within a pH range of 4.0 to 8.0. Spores of Fuligo septica germinated from pH 2.0 to 10.0. Optimum for all species ranged from 4.5 to 7.0, with some germinating better near 4.5 and others near 7.0. The spores of Urocystis occulta germinated between pH 5.0 and 8.9, with the optimum at 6.8 (Ling, 1940). This optimum is higher than those for most fungi. Kauffman (1934) found the range for spore germination of several species of Basidiomycetes (Agaricaceae and Nidulariaceae) to be pH 5.0 to 8.5 with the optimum near 7.5. It is interesting that Doran (1922) in his review of spore germination makes no mention of acidity as a factor. It would appear that acidity is of more or less importance as a modifying factor, even though it is seldom a limiting factor for spore germination. This may explain, at least in part, the fact that we often find abundant ungerminated spores in fruiting liquid cultures. Some fungi sporulate only in neutral or alkaline media, which, in general, are not favorable to spore germination. NUTRIENTS AND STIMULANTS The constituents of the substrate are known to influence spore germina- tion of some species of fungi. Some species germinate well in distilled or tap water, while others require certain special nutrients such as sugar, salts, or even a particular nitrogen source. No one medium has been found which will allow good germination of all fungi, although certain natural media, such as beet or bean decoction and soil infusion, seem to favor germination in a large number of fungi. When such media con- taining natural products are used, it is difficult to determine whether the higher percentage of spore germination is due to the nutrients or to some stimulant which is not used in the metabolism of the fungus. Duggar w^as one of the foremost American workers interested in spore germination as a primary subject of experimentation. Prior to his work, most of the study on spore germination was only incidental to other problems. Duggar (1901) demonstrated that species differ in their nutrient requirements for germination by placing spores in water, bean decoction, nutrient-salt solution, and cane-sugar solution. A portion of his data showing the percentage of germination after 15 hr. is given in Table 59. Among some of the compounds Duggar found to influence sporulation of Aspergillus flavus and A. niger were varying amounts of peptone, ammonium nitrate, and magnesium sulfate. Ammonium nitrate at a 362 PHWSIOLOGY OF THE FUNGI particular concentration gave abundant germination of A . flavus but had no effect upon ^4. niger. Brefeld (1905) was perhaps the first to observe the germination of the spores of various smuts in culture. He noted that the spores germinated poorly or not at all in water, while excellent germination occurred in nutrient solutions (probably dung infusion). Brefeld expressed surprise at the vigorous saprophytic development which followed, especially since the species had previously been known only as obligate parasites. More recently it was noted that pretreatment with dung infusion markedly stimulated germination of spores of Ustilago striiformis (Cheo Table 59. Percentage of Spore Germination after 15 Hours (Duggar, Botan. Gaz. 31, 1901.) Spores of Aspergillus niger Penicillium glaucum Monilia fructigena Mucor spinosus Phycomyces nitens Coprinus jimetarius C. comatus C. micaceus Uromyces caryophyllinus Water 0 0 75 0 0 0 0 0 100 Bean decoction 100 100 100 100 100 5-10 0 100 75 Xutrient-salt solution 100 100 100 100 100 0 0 0 Sucrose solution 75 1 100 1 2-10 0 0 0 100 and Leach, 1950). Untreated spores in distilled water germinated only after 5 to 8 days, and the total germination was less than 1 per cent. Spores soaked in a concentrated horse-dung infusion for 15 days or more, then placed in distilled water, germinated within 5 hr., with a total germination of 50 per cent or higher. The exposure to the dung infusion is believed to increase the permeability of the spore wall, allowing the more rapid absorption of water. It might also be pointed out that the dung infusion evidently contains substances which prevent spore germina- tion until highly diluted or removed entirely. Although the spores of the Myxomycetes germinate in distilled water, the percentage may be greater in weak decoctions of the natural sub- strate, such as rooting wood, bark, leaves, or humus (Smart, 1937). Similarly, the conidia of PhyUosticia solitaria germinate more profusely in apple-bark decoction and potato-dextrose broth than in distilled water (Burgert, 1934). While it is possible that increased spore germination is due primarily to some stimulating substance, it seems likely that certain nutrients are also involved. The conidia of Glomerella cingulata apparently have special nutritional requirements for germination. There was little or no germination in SPORE GERMINATION 363 distilled water and in dextrose solution lacking minerals (Lin, 1945). From his experiments involving various inorganic compounds, Lin concluded that carbon, magnesium, nitrogen, and phosphorus, are required (Table 60). The need for sulfur was not so evident as that for the other elements, and sulfur was not essential. The minimum require- ments of nitrogen and phosphorus were calculated to be of the order of 10"' Mg per spore. No evidence was found that an external supply of any organic substance, other than sugar, is necessary for spore germination. Table 60. The Essentiality of Various Ions for the Germination of the CoNiDiA OF GlomereUa cingulata (Lin, Am. Jour. Botany 32, 1945.) Chemical substance applied* Element lacking Germination, % None (redistilled water) dnrosR Carbon and minerals Minerals None None Nitrogen Potassium Phosphorus Sulfur Magnesium Carbon 0.0 0.0 Glucose, KNO3, KH2PO4, MgS04 Glucose, NH4CI, KH2PO4, MgS04 Glucose, KCl, KH.,P04, MgS04 Glucose, NaNOs, NaH,P04, .MgS04 Glucose, KNO3, KCl, MgS04 Glucose, KNO3, KH2PO4, MgCh Glucose, KNO3, KH.POj, ^aSO, KXO,, KH2PO4, MgS04 80.4 92.8 3.9 84.1 1.5 79.3 0.9 0.7 * In all cases, the concentration of glucose is 0.01 per cent, that of each of the mineral salts 1.0 milli- mole. The constituents of the medium may modify the effects of pH on spore germination. This is illustrated in Fig. 75 by the germination of Lenzites saepiaria on 2 per cent bacto-peptone, in sucrose-nitrate (Czapek's) solution, and in beet decoction (Webb, 1921). Emerson (1948) showed that D-xylose as a carbon source gave a high percentage of germination of ascospores of Neurospora crassa without heat treatment. Xylose was more effective when autoclaved than when filtered. This was believed to be due to the slight conversion to furfural, which was also shown to be active in increasing spore germination. From this brief discussion it is evident that little is known about the effects of nutrition upon spore germination. This is no doubt due, in part, to the lack of planned experimental work along this line. Many of the favorable effects of natural products may in fact be due to the presence of stimulants rather than to the nutrients. At the present time we have no conclusive evidence that spores require an external source of vitamins for germination. In the light of the recent discovery of Ryan 364 PHYSIOLOGY OF THE FUNGI r A 1 1 \ \ \ ■^V ■s \ V^ ■ J \ 1 '; / • / c' \ ^ \ I J/ / X V, \ (1948) that the amino acids leucine, lysine, and proline favored spore germination in mutants of Neurospora deficient for those amino acids, it also seems likely that spore germination in certain vitamin-deficient fungi may be aided by the addition of the vitamins in question. A careful study of the effects of vitamins is needed. The spores of some fungi, such as Botrytis cinerca, germinate much better Avhen in contact with plant tissue than in distilled water (Brown, 1922). It was concluded that certain substances diffuse out of the host plant into the infection drop containing the spores and stimulate germina- tion and infection. Leach (1923) believes that a similar situation may 100 80 c ,o o ■| 60 a> o> § 40 I 20 » 23456789 pH of medium Fig. 75. The effect of the pH and kind of medium on the percentage of germination of spores of Lenzitcs sacpiaria at 20 to 23°C. A, in sugar-beet decoction; B, in 2 per cent bacto-peptone sohition; C, in Czapek's full nutrient solution. (Redrawn from Webb, Ann. Missouri Botan. Garden 8: 325-327, 1921.) exist with Colletotrichum Undemuthianum. The spores of this fungus germinated poorly in distilled water alone, but distilled water plus a piece of fresh bean tissue gave a high percentage of germination. Fresh bean juice was equally effective, but boiled bean decoction did not stimu- late germination. However, green-bean agar made from a similar decoc- tion gave excellent germination, as did potato-dextrose agar. These results led Leach to conclude that two distinct stimulating factors may be involved. A portion of Leach's data is summarized in Table 61. Some know^n stimulants may eliminate the need for certain factors ordinarily supplied by natural media for the germination of spores of Phycomijces (Robbins et al., 1942). Germination of spores was about 12 per cent or less on mineral-dextrose agar with thiamine. The addition of an extract of potatoes, or of other natural products, of hypoxanthine, acetate, or some other organic acids increased germination to nearly 100 per cent. Treatment of spores with aqueous pyridine had the same favorable effect. These authors believe that certain factors (called Z factors) are essential in spore germination. One of these (factor Zi) SPORE GERMINATION 365 has been identified as hypoxanthine, while the identity of factor Z2 i» still unknown. An explanation of the effects of these stimuli is given by these authors: The dormant spores are considered to lack sufficient available Z factors for germination. The extracts of natural products or the Z factors furnished in the medium supply this deficiency, which may also be met by treatment with heat, cold, acetate or pyridine. These treatments are thought to change the Z factors in the spores from an unavailable to an available form. The effects of certain gases and volatile compounds upon germination have also been demonstrated. It has been observed that spores of num- erous fungi germinate better in a container in which some living plant part is also present. This was demonstrated for Basisporium gallarum by Durrell (1925), who also found that the introduction of carbon dioxide T.^ELE 61. The Effect of Various Media and Plant Tissues on Spore Germina- tion OF Colletotrichuni lindemuthianum (Leach, Minn. Agr. Expt. Sta. Bull. 14, 1923.) Medium Germination, % Distilled water 3-6 Sucrose-nitrate (Czapek's) solution 5-11 Sucrose-nitrate (Czapek's) solution plus bean decoction 10 Bean decoction 8 Distilled water plus fresh bean tissue 83-95 Distilled water plus sunflower tissue 5 Distilled water plus wheat tissue 12 Distilled water plus corn tissue 10 Distilled water plus tomato tissue 2 Sucrose-nitrate (Czapek's) solution plus bean tissue 95 Green-bean agar 97 Potato-glucose agar 98 into the container enclosing the spores gave the same increase in germina- tion. The same effect was demonstrated for Ustilago zeae (Platz et at., 1927). An atmosphere containing 15 per cent carbon dioxide was found to be optimum for spore germination. Such a condition gave a pH of the medivmi from 4.9 to 5.6. These authors conclude that the stimulating effect is apparently due to "a definite action of carbonic acid." Is it possible that this is an example of heterotrophic utilization of carbon dioxide? While the release of carbon dioxide into the atmosphere by various living plant parts may explain the stimulation of spore germination in many cases, the presence of carbon dioxide alone will not explain certain results obtained by some workers. For instance, spore germination of Botrijtis cinerea was stimulated by the presence of living tissues of apples or leaves of Ruta or Eucalyptus in the same container, while tissues of potato tuber and onion scales inhibited germination (Brown, 1922). 366 PHYSIOLOGY OF THE FUNGI Distillates of these leaves increased germination four to ten times. Ethyl acetate likewise gave similar results. The possibility of specific activity was suggested by the fact that apple tissues distinctly stimulated germina- tion of B. cinerea spores, while they inhibited germination of spores of Colletotrichum lindemuthianum. The stimulation was greater with old spores. Presoaking and the subsequent addition of a stimulating volatile agent gave optimum germination of Urocystis tritici spores (Noble, 1923). The expressed sap of wheat placed in the same container with germinating spores, but in separate dishes, proved to be a good stimulating agent. Uninjured seedlings of certain nonsusceptible hosts likewise stimulated spore germination. Benzaldehyde, salicylaldehyde, butyric acid, and acetone in certain concentrations stimulated germination of presoaked spores. Noble believed that presoaking increased permeability of the spore and allowed the more rapid intake of the stimulatory volatile sub- stance, which increased the permeability of the protoplasmic membrane by changing its physical condition. Likewise, a solution of benzaldehyde (3/2,000,000) stimulated germina- tion of Urocystis occulta spores, which germinated very poorly in water (Ling, 1940). Ethyl alcohol stimulated spore germination in Aspergillus flavus; methyl alcohol was slower and less effective (Duggar, 1901). A. niger was stimulated by oxalic acid, whereas .4. fiavus was not. It is understood that the stimulatory power of these chemicals depends upon the concentration. An interesting situation exists in the germination response of some spores to the presence of other fungi, or even to the medium in which other fungi have grown. The few experiments conducted along this line suggest that the constant association with other organisms may be highly beneficial to spore germination as well as subsequent growth of some fungi in nature. The germination of a number of species of Myxomycetes was increased by the addition of the filtrate of a medium in which spores had previously been germinated (Smart, 1937). Smart calls the stimulatory factor an "autocatalytic agent." A portion of Smart's data is presented in Table 62. Fries (1941, 1943) obtained almost phenomenal results with spores of a number of Hymenomycetes, which previously had germinated poorly or not at all, by sowing the spores on malt agar with living cultures of Torulopsis sanguinea. Spores of ten species of Tricholoma, which ger- minated only with difficulty without the yeast, were found to germinate readily in its presence. One species of Tricholoma gave only negative results. In Amanita mappa, A. porphyria, and A. rubescens germination occurred only when Torulopsis was present. Germination of two other SPORE GERMINATION 367 species of Amanita was considerably improved by the presence of the yeast. None of the seven species of Boletus germinated on malt agar without the yeast. On the same medium and in the presence of Torulopsis sanguinca, germination was obtained with spores of B. bovinus, B. elegans, B. flavidus, B. granulatus, B. luteus, B. variegatus, and B. viscidus. Some germination of Boletus spores was also obtained in the presence of living colonies of certain other fungi, but none was so effective as Torulopsis. Spores of certain other fungi {Hijdnum repandum, H. imhricatum, Craterel- lus lutescens, Lycoperdon umhrinum, L. echinatum., L. nigrescens, L. pra- tense, L. pyriforme, and Scleroderma aurantium) germinated in Fries's Table 62. Germination of Single Myxomycete Spores (Smart, Aryi. Jour. Botany 24, 1937.) ' Number of spores germinating Species Lot 1 (10 spores) (fresh medium) Lot 2 (10 spores) (previous germination medium) Vulino sevtica, 9 after 3 hr. 6 after 3 days 3 after 2 days 4 after 1 day 0 in 2 weeks 0 in 2 weeks 3 after 2 days 6 after 6 days 2 after 18 days 9 after 7 days 10 in 45 min. Physarum polycephahini Stemonitis fusca S ciTifero, 8 in 15 hr. 9 in 1 day 8 in 8 hr. Enteridium rozeanum Reticularia lycoperdon Lycogala epidendrum Arcyria denudata Dictydium cancellaium Physarum cinereum 10 in 30 min. 8 in 15 min. 8 in 3 hr. G in 6 days 2 after 18 days 9 in 6 days experiments only in the presence of T. sanguinea. He also tested the effects of mycelial extracts on spore germination and found that extracts of certain species of Boletus stimulated germination of spores of the same species. Many of the fungi studied by Fries are believed to be mycorhizal and may require the presence of a special set of conditions, perhaps the roots of certain plants (or conditions which simulate their presence), before germination will occur. The time required for a spore to germinate after being subjected to favorable conditions is a reflection of the interaction and relative impor- tance of all the various influencing factors. The nearer all these factors are to the optimum, the shorter will be the time required for germination. Time is an important factor for the subsequent infection of the host. In nature the near-optimum environmental conditions, principally tempera- ture and moisture, may persist for bvit a short time, and a change in but one of these factors may inhibit spore germination. 3G8 PHYSIOLOGY OF THE FUNGI LONGEVITY OF SPORES The length of Hfe of spores is usually measured by their ability to germinate after various periods of time. It is affected by environmental conditions, principally temperature and moisture. The greatest period of longevity reported for fungus spores appears to be among the Myx- omycetes. Smith (1929) succeeded in germinating spores from herbarium specimens of Myxomycetes 5 to 32 years after they were collected. A few of the common species whose spores germinated after approximately 30 years are Physarum cinereum, Fuligo septica, Hemiirichia clavata, and Stemonitis ferruginea. Smut spores also have a long period of viability (Lowther, 1950). Spores of Aspergillus orijzae germinated after 22 years in a sealed tube at room temperature (McCrea, 1923). In contrast to long periods of longevity, some fungus spores die very soon after they are liberated. The sporidia of Cronartium rihicola lived less than 10 min. at room temperature with a humidity of 90 per cent (Spaulding, cited by Doran, 1922). Sporidia of Gymno sporangium juniperi-virginianae lived no longer than 6 days in dry air. Eight weeks is reported as the maximum longevity of aeciospores of C. rihicola, with only 5 per cent germination after 7 weeks. In general, aeciospores of the rust fungi remain viable about 50 per cent longer than the urediospores, whose average longevity ranged from 30 to 60 days (Doran, 1922). Other factors have been reported to influence longevity of spores. Ascospores of Endothia parasitica remained viable for a year when dried in the bark, but when removed from the bark, they lost the ability to germinate within 5 months (Anderson and Rankin, 1914). Similarly, conidia in dry spore horns retained viability for at least a year, but when placed in water, separated, and then dried, the time was less than 1 month. It seems likely that one of the functions of the gelatinous matrix of the conidia of certain fungi, such as Gloeosporium, Colletotrichum, and Cytospora, is to increase the longevity of the spores through its water- holding capacity. Light is apparently only of minor importance as a factor influencing longevity. No doubt ultraviolet light in nature plays an important part in reducing the period of viability and even in killing many of the hyaline spores. Spores having dark walls are protected somewhat against the penetration of the ultraviolet rays. SUMMARY Spore germination represents a change from an inactive to an active phase in the life cycle of a fungus. Since it involves the first stages of growth, it is reasonable to expect that many of the factors which influence vegetative growth also affect spore germination. On the other hand, the spore, being a resting cell, may contain stored materials not usually SPORE GERMINATION 369 present in appreciable quantities in vegetative cells. Since the metabolic activity of a resting spore is at a minimum in contrast with that of actively- growing vegetative cells, the internal responses to the environmental factors may be quite different. The variability of the needs of spores of different fungi for germination is adequately illustrated in the literature. Certain general conditions are essential for all spores, while some require a special set of conditions. Water is essential to activate certain enzyme systems, to initiate other internal chemical changes, and to increase the volume of the germinating spore. WTien the temperature is near the optimum, the enzymatic activity and the rate of spore germination are increased. The supply of oxygen must be adequate to meet the demands of the greatly increased rate of respiration. The acidity of the substrate must be favorable. Variability in the period of viability of spores is striking, but longevity is greatly influenced by the environment. Much information is yet to be gained regarding the longevity of spores, particularly of the plant pathogens. Certain special conditions are required for germination of some spores. These may act as a stimulant in breaking dormancy or may supply needed nutrients. The effects of other living organisms, or even of the substrate upon which they have grown, are of particular interest, for such associa- tion is the usual condition under which germination occurs in nature One might suppose that the secretions of certain plants would exert a selective action on spore germination and affect the pathogenicity of certain fungi, but evidence on this point is lacking. The production of short germ tubes by spores of some species of Ery- siphe in an absolutely dry atmosphere is unusual. If this is to be con- sidered as true germination, it must represent a unique method among fungi. The Erysiphales, however, are excellent examples of fungi whose spores germinate in atmospheres of lower relative humidity than most fungi can endure. Under the changing conditions of nature, the period of time during which a factor is active is of utmost importance. Germination is the result of the action of all the influencing factors operating at the same time. Most of these factors vary in intensity or concentration, so that the combined optima of all factors are seldom, if ever, reached at any given time in nature. As a result, an extremely low percentage of the spores formed by a fungus ever germinate, while still fewer give rise to extensive mycelium. REFERENCES Anderson, P. J., and W. H. Rankin: Endothia canker of chestnut, Cornell Univ. Agr. Expt. Sta. Bull. 347, 1914. Bonner, J. T. : A study of the temperature and humidity requirements of Aspergiltus niger, Mycologia 40: 728-738, 1948- 370 PHYSIOLOGY OF THE FUNGI Brefeld, O.: Die Brandpilze IV. in Untersuchungen aus dem Gesammtgebiete der Mykologic, Heft 13, Muenster, Heinrich Schoningh, 1905. *Brodie, H. J.: Further investigations on the mechanism of germination of the conidia of various species of powdery mildew at low humidity, Can. Jour. Research 23: 198-211, 1945. Brodie, n. J., and C. C. Neufeld: The development and structure of the conidia of Enjsiphe polygoni DC. and their germination at low humidity. Can. Jour. Research 20: 41-61, 1942. *Brown, W. : Studies in the physiology of parasitism. IX. The effect on the germi- nation of fungal spores of volatile substances arising from plant tissues, Ann. Botany 36: 285-300, 1922. BuRGERT, I. A.: Some factors influencing germination of spores of Phyllosticta solitaria, Phytopathology 24: 384-396, 1934. *Chbo, p. C, and J. G. Leach: The stimulating effect of dung infusion on the germi- nation of spores of Ustilago striiformis, Phytopathology 40 : 584-589, 1950. *Clayton, C. N.: The germination of fungous spores in relation to controlled humid- ity, Phytopathology 32: 921-934, 1942. Dodge, B. O.: Methods of culture and morphology of the archicarp in certain species of the Ascobolaceae, Bull. Torrey Boian. Club 39: 139-197, 1912. DoRAN, W. L. : Effect of external and internal factors on the germination of fungous spores, Bull. Torrey Botan. Club 49: 313-336, 1922. DuGGAR, B. M.: Physiological studies with special reference to germination of certain fungous spores, Botan. Gaz. 31: 38-66, 1901. DuRRELL, L. W. : Basisporium dry rot of corn, Iowa Agr. Expt. Sta. Bull. 84, 1925. Emerson, M. A.: Chemical activation of ascospore germination in Xeurospora crassa, Jour. Bad. 55 : 327-330, 1948. Fries, N.: Ueber die Sporenkeimung bei einigen Gasterromyceten und mykor- rhizabildenen Hymenomyceten, Arch. Mikrobiol. 12: 266-284, 1941. Fries, N.: Untersuchungen iiber Sporenkeimung und Mycelentwicklung boden- bewohnender Hymenomj'ceten, Symholae Botan. Upsaliensis 6: 1-81, 1943. Gardner, M. W. : Anthracnose of cucurbits, U.S. Dept. Agr. Bull. 727, 1918. GoDDARD, D. R. : The reversible heat activation inducing germination and increased respiration in the ascospores of Neurospora tetrasperma, Jour. Gen. Physiol. 19 : 45-60, 1935. GoDDARD, D. R., and P. E. Smith: Respiratory block in the dormant spores of Neurospora tetrasperma, Plant Physiol. 13: 241-264, 1938. *GoTTLiEB, D.: The physiology of spore germination in fungi, Botan. Rev. 16 : 229-257, 1950. Jones, E. S. : Influence of temperature, moisture and oxygen on spore germination of Ustilago avenae, Jour. Agr. Research 24: 577-591, 1923. Kauffman, F. H. O. : Studies on the germination of the spores of certain Basidio- mycetes, Botan. Gaz. 96 : 282-297, 1934. Leach, J. G.: The parasitism of Colletotrichum lindemuthianum, Minn. Agr. Expt. Sta. Bull. 14, 1923. *LiN, C. K.: Nutrient requirements in the germination of the conidia of Glomerella cingulata, Am. Jour. Botany 32 : 296-298, 1945. Ling, L. : Factors affecting spore germination and growth of Urocystis occulta in culture. Phytopathology 30: 579-591, 1940. Lowther, C. v.: Chlamydospore germination in physiologic races of Tilletia caries and Tilletia foetida, Phytopathology 40 : 590-603, 1950. McCrea, a.: Spores of Aspergillus oryzae alive after 22 years stored in test tube. Science (N.S.) 58: 426, 1923. SPORE GERMINATION 371 Noble, R. J.: Studios on the parasitism of Urocystis tritici Koern, the organism causing flag smut of wheat, Jour. Ayr. Research 27 : 451-489, 1924. Platz, G. a., L. W. Durrell, and M. E. Howe: Effect of carbon dioxide upon the germination of chlamydosporcs of Ustilago zeae (Beckm.) Ung., Jour. Agr. Research 34 : 137-147, 1927. RoBBiNS, W. J., V. W. Kavanagh, and F. Kavanagh: Growth substances and dormancy of spores of Phycomyces, Botan. Gaz. 104 : 224-242, 1942. Ryan, F. J.: The appHcation of Neurospora to bioassay, Fed. Proc. 3 : 365-369, 1946. *Ryan, F. J.: The germination of conidia from biochemical mutants oi Neurospora, Am. Jour. Botany 35: 497-503, 1948. Smart, R. F.: Influence of certain external factors on spore germination in the Myxomycetes, Am. Jour. Botany 24: 145-159, 1937. Smith, E. C: The longevity of Alyxomycete spores, Mycologia 21: 321-323, 1929. Uppal, B, N.: Relation of oxygen to spore germination in some species of Perono- sporales, Phytopathology 16 : 285-292, 1926. Walker, J. C., and F. L. Wellman: Relation of temperature to spore germination and growth of Urocystis cepulae, Jour. Agr. Research 32: 133-146, 1926. ^Webb, R. W.: Studies in the physiology of fungi. XV. Germination of the spores of certain fungi in relation to hydrogen-ion concentration, Ann. Missouri Botan. Gardens: 282-341, 1921. Wolf, F. A., and F. T. Wolf: The Fungi, Vol. II, John Wiley & Sons, Inc., New York, 1947. Yarwood, C. E.: The tolerance of Erysiphe polygoni and certain other powdery mildews to low humidity, Phytopathology 26 : 845-859, 1936. CHAPTER 17 THE PHYSIOLOGY OF PARASITISM AND RESISTANCE A discussion of parasitism occupies an important position in any treatise on the physiology of fungi, particularly for those students who are interested in plant diseases or the fungi which cause them. This phase of study offers many challenging unsolved problems. Parasitism involves primarily two living organisms, the parasite, whose actions are offensive, and the host, whose reactions are defensive. If the defenses of the host plant, either before or after penetration by the parasite, are successful, the plant is resistant; if not, it is susceptible. To be successful, a parasite must find the nutritional and environmental conditions favorable for its development. If even a single important factor is unfavorable to the parasite, the fungus may fail to establish a parasitic relationship with its proposed host. Such factors may exert their influence either before or after penetration by the fungus. Environmental factors acting before penetration may in reality bring about an escape from a disease rather than true resistance to it. The present discussion is divided into three main parts: (1) penetra- tion; (2) parasitism, the action of the parasite in becoming established and obtaining its food ; (3) resistance of the host to penetration or against the parasite after penetration. The comprehensive reviews of the physiology of the host-parasite relationship given by Brown (1936, 19-48) should be read by all students. Similar reference is made to Arthur et al. (1929), who give an excellent discussion of the parasitic relations of the rusts, and to the treatise of Gaumann (1946, 1950) on the principles of plant infection, PENETRATION A parasite may gain entrance into the host (1) through the natural openings, such as stomata or lenticels, (2) by direct penetration through the uninjured epidermis, or (3) through wounds. Through stomata. Viable spores may fall upon a host plant and pro- duce germ tubes, which by chance grow over or near stomata. The outer walls of the epidermal cells of aerial plant parts are covered with cutin, which is somewhat resistant to penetration by some fungi. The germ tube which enters through a stoma may then be favored by the moist atmosphere in the substomatal cavity. In some cases, the unspecialized hyphae may penetrate the host cells; in other fungi, haustoria, which 372 PARASITISM AND RESISTANCE 373 arise from the intercellular mycelium, penetrate the host cells and absorb food. Water vapor has been suggested as the stimulus which causes the germ tube to turn inward and enter a stoma. This, however, cannot be the case with zoospores which are immersed in water and which have been noted to cluster around stomata. The fungi which normally enter the host plant through stomata include the cereal rusts (aeciospore and urediospore stages), Cercospora heticola, Phytophthora infestans (zoospore stage), the Peronosporales, Albugo Candida, and others. The cereal rusts have received a great deal of attention in resistance studies. It has been reported (Hart, 1929) that Puccinia graminis apparentl}^ requires the open stomata of wheat plant for penetration. On the other hand, Caldwell and Stone (1936) have shown that the germ tubes of Puccinia triticina are able to force their way between the guard cells of closed stomata of wheat leaves. A germ tube from a urediospore may start to enter an open stoma, but as it forms an appressorium, the stoma closes. Further penetration is accomplished between the guard cells by a slender hypha. Allen (192G) believes that the appressorium probably secretes some toxin which harms or even kills the guard cells, causing the stoma to close. Caldwell and Stone, however, do not believe that this injury to the guard cells is necessary for entry of germ tubes. The appressorium seems to function as a special organ to apply the pres- sure needed for the forced entry between the closed guard cells. Penetration through lenticels more often occurs in the underground parts of the host which are in a more or less moist situation. Potato tubers may become infected by Actinomyces scabies and by germ tubes from sporangia of Phytophthora infestans, chiefly through the lenticels. Direct penetration. A large number of fungi are capable of penetrating the unbroken epidermis of a plant, directly through the cutinized outer walls. The spore may germinate on the surface of the plant in a drop of water. The germ tube grows over the epidermis and by some stimulus is caused to turn inward and penetrate the cell. Brown (1922) demon- strated that there is a certain amount of exosmosis of materials from host tissue into a drop of liquid on the surface. In some cases this may lead to a chemotropic response by the fungus. However, in most cases the stimulus of contact is believed to initiate appressorial formation and penetration. The formation of appressoria is common among many fungi when the germ tubes come in contact with the epidermal cells. The fact that the appressoria are often formed on a glass slide is further evidence that their formation is in response to contact with a solid sur- face. Appressoria are usually bulb-like or disk-like in shape and are believed to serve as an adhesive disk against which the slender infection hypha may push in penetrating the cell wall. Brown (1915, 1922) presents evidence that the host cells are not killed by Botrytis cinerea, 374 PHYSIOLOGY OF THE FUNGI Sclcrotinia sclerotiorum, and Collctoirichum lindemuthianum until after the fungus penetrates the cuticle of the host. In other words, there is little or no diffusion of the toxic materials through the cuticle. Direct penetra- tion through cutinized walls is believed to be entirely by mechanical pressure, since no cutin-dissolving enzyme has been demonstrated in the fungi. The rhizomorphs of Armillaria mellea usually gain entrance directly through the sound cork layer of comparatively old roots (Thomas, 1934). Penetration is believed to be accomplished partly by mechanical pressure and partly by chemical means. There is evidence that a suberin-dis- solving enzyme aids in the destruction of some of the cork cells. Some fungi may enter the same host by more than one method. Fusarium lint may enter through young epidermal cells of the root, root hairs, stomata of seedlings, and perhaps through wounds. The penetration of noncutinized cell walls may be either by mechanical pressure or by the dissolving action of enzymes secreted by the fungus. Hawkins and Harvey (1919) concluded that the hyphae of Pythium debaryanum penetrated the cell walls of susceptible potato tubers by mechanical pressure, and that the resistant varieties in general showed greater resistance to mechanical puncture. They found no evidence of cellulases which might aid in penetration by dissolving the cellulose cell wall. Using cane sugar as the plasmolyzing solution, they found that the hyphae of P. debaryanum were capable of exerting as high as 54 atm. osmotic pressure. These hyphae would have a strong tendency to absorb water, and as a result greater internal pressure would be exerted against the hyphal wall. Apparently the hyphal wall is capable of withstanding this pressure at all points except its tip, where growth occurs. The pressure exerted by the growing tip is believed to be sufficient to cause penetration of the host cell wall. By direct microscopic examination Hawkins and Harvey observed that, just after the hyphal tip came in contact with the host cell wall, it formed a swelling, back of which a bend developed. This was followed by penetration of the wall by a small tube. Penetration through noncutinized cell walls by chemical means has been described for Spongospora suhterranea by Kunkel (1915). It seems likely that other nonfilamentous fungi penetrate cell walls in the same way. Likewise, wood rot fungi penetrate the cellulose and lignified cell walls by enzymatic action, as evidenced by the boreholes in decaying wood. It may be significant that the hyphal walls of Pythium, as well as of other Oomycetes, contain cellulose, while the hyphal walls of other fungi are composed principally of chitin, which would not be acted upon by cellulases. It must be emphasized that penetration of the host in itself does not necessarily lead to the establishment of the fungus in the host and the PARASITISM AND RESISTANCE 375 production of a disease. In some cases it is known that a fungus may enter resistant or immune plants, as well as susceptible ones, but find the conditions unfavorable for its establishment and further development. Through wounds. A number of fungi apparently are unable to pene- trate a healthy plant except through wounds. These may be insect wounds, broken branches of trees, broken roots, etc. In addition, some fungi which are capable of entering the host by other means may also penetrate through wounds. Phymatotrichum omnivorum, the cause of numerous root rots, commonly enters roots through wounds, although these are not necessary. Fusarium, causing dry rot of potato, apparently enters the tubers only after they have been wounded. Likewise most of the wood-rotting Basidiomycetes enter the host only through wounds, principally at broken or dead branches and at pruning or lightning and fire scars. Here, the air-borne basidiospores must find suitable moisture for germination and for penetration of the wood. Endothia parasitica is said to enter the chestnut tree only through wounds that extend through the corky layer. Ceratostomella ulmi is transmitted by the European bark beetle, which introduces the spores into its feeding wounds. Bruises and wounds of fruits and vegetables are common ports of entry for numer- ous rot-producing fungi, such as Rhizopus nigricans on sweet potato. Monilinia fructicola on stone fruits, Penicillium expansum on apple, and P. italic^im. and P. digitatum on citrus fruits. PARASITISM A discussion of the action of the parasite after it enters the host is so closely correlated with the defense of the host that it is difficult to discuss each topic separately. For the sake of convenience, however, it seems desirable to discuss some of the outstanding effects of fungi upon their hosts and the methods by which the parasites obtain their food under a separate heading of parasitism. Parasitism in plants. Parasitism may begin as soon as a fungus hypha enters the host. The primary consideration is the securing of suitable nutrients and water by the fungus. This may be accomplished by two general methods, (1) by killing the cells of the host and obtaining food from the dead cells, or (2) by establishing a close nutritional relationship with the living host cells and absorbing the soluble nutrients without causing necrosis. The fungi falling in the first group are the destructive parasites, while those belonging to the second group have been called the balanced parasites (Bessey, 1935). The latter group includes those fungi known at present as obligate parasites (such as the Uredinales, Erysiphales, and Peronosporaceae), and some other fungi (such as the Ustilaginalesand Taphrina) which in their hosts obtain food only from living cells. The destructive parasites, as a whole, are strong producers of enzymet; 376 PHYSIOLOGY OF THE FUNGI and toxins but may be weak in mechanical action. Some of these cause rapid rots of fruits or vegetables but are unable to penetrate the unbroken epidermis and must depend on wounds for their entrance. Others, which are seldom, if ever, found as pathogens in nature, may cause rot when artificially inoculated into succulent plant tissues. Rotting of the tissue is due to two distinct effects of the fungus on the host: (1) death of the cells, and (2) dissolution of the middle lamellae. The separation of the cells is due to the action of the enzymes proto- pectinase, pectinase, and pectase on the middle lamella. These three enzymes are often collectively referred to as pectinase. There is some evidence that pectinase may also cause a change in permeability of the cell membranes and the death of the cells, but it is possible that some other toxic substance may be closely associated with pectinase. How- ever, no such substance has been isolated. Extracts of rotted tissues have been shown to cause the same effects as the fungi themselves. These effects are described by De Bary (1886) for Sclerotinia sclerotiorum and by Brown (1915) for Botrytis cinerea. Higgins (1927) believes that oxalic acid produced by Sclerotium rolfsii is the principal agent of destruc- tion. The death of the host cells well in advance of the invading hyphae indicates rapid diffusion of the toxic substance in the case of fungi produc- ing soft rot. Brown (1948) believes that the enzyme pectinase acts as a cytolytic toxin. For a discussion of the identity of enzymes and toxins of species of Clostridium, see Smith (1949). Thatcher (1942) has shown that B. cinerea and S. sclerotiorum cause a fourfold increase in the permeability to water of the host cells just beyond the discolored necrotic zone. Some substance other than pecti- nase may bring about this change in permeability and be a contributing factor to the "action in advance" of many fungi. PhytophtJwra infestans caused a change in permeability in host cells beyond the extent of the hyphae which penetrated the living tissue. The identity of the substance causing a change in permeability is unknown, but it is likely a weak toxin or an enzyme which alters the structure or activity of the plasma mem- brane. The increase in permeability may concern water alone or both nutrients and water. An osmotic pressure higher in the fungus cells than in the surrounding host cells is apparently characteristic of the host-parasite relationship (Table 63) . This is necessary before the parasite can absorb water from the host cells. The production of pectinase and its activity under different conditions were studied by Vasudeva (1930) and Chona (1932), who showed that the amount produced by Botrytis allii varied with the medium in which the fungus was grown. B. allii did not secrete a demonstrable amount of pectinase when grown on apple extract, but when asparagine, potassium PARASITISM AND RESISTANCE 377 nitrate, or ammonium sulfate was added to the apple extract, there was a decided increase in the amount produced. This is not surprising, for there are numerous reports that the available nutrients and the pH of the culture medium affect both the kind and amount of metabolic products of a fungus. The activity of the enzymes produced by a pathogen varies with the conditions of the host cells. It seems probable that the inhibition of the fungus enzymes by the host cells is an important factor in resistance. Klotz (1927) proposed this hypothesis to explain the greater resistance of sour orange and the greater susceptibility of lemon to Pythiacystis citrophthora and Phomopsis calif or nica, the causes of certain bark diseases. Table 63. The Osmotic Pressures of Host and Parasite (Thatcher, Can. Jour. Research 20, 1942.) Fungus Ave. osmotic pressure, atm. Host Ave. osmotic pressure, atm. Uromyces fabae, germ tubes . . . haustoria 44.25 21.9 18.6 29.8 23.5 18.9 18.0 17.4 15.5 18.1 41.3 Pisum sativum, leaf petiole Dianthus, leaf base 9.15 10.1 U. caryophyUinus, haustoria. . . Botrytis cinerea, hyphae Sclerotinia sderoliorum, hyphae. Puccinia graminis, haustoria (race 21) 11.2 Apium graveolens, petiole A. graveolens, petiole Mindum wheat, leaf Brassica, leaf Solanum tuberosum, tuber netiole 8.3 13.4 9.4 Erysiphe polygoni, hyphae Phytophihora infestans, hj^phae (aerial) 10.6 10.6 hyphae (intercellular) sporangia 8.9 Brassica, root Phoma lingam, hyphae 11.3 The greater pathogenic action of a destructive fungus occurs in the host whose cells are favorable for the activity of the enzymes of the fungus. Further evidence of enzyme inhibition of certain plant tissues was presented by Chona (1932), who studied the rotting action of B. cinerea, the cause of a soft rot of various plant tissues, and Pythium sp., a rot producer of potato tubers. Vigorous germination of spores of Botrytis and even some sporulation took place in artificial wounds in potato tubers, but no decomposition followed. The pectinase produced by B. cinerea was active against apple tissue, but the presence of potato tissue inhibited its activity. It was then found that the mineral salts, particu- larly KH2PO4 and MgS04, in the potato were the inhibiting factors. On the other hand, Pythium spores germinated well on apple tissue but failed 378 PHYSIOLOGY OF THE FUNGI to rot it. The inhibition in this case was traced to the mahc acid in the apple. The pectinase produced by Pythium was most active in an alkahne medium, near pH 8.0, while that of B. cinerea was more active in an acid medium, at pH 5.0 to 5.5. In contrast with the destructive fungi which rot the host tissue are those which cause wilting and certain types of necrosis without disintegra- tion of the host cells. These fungi produce little or no pectinase. Some common fungi which cause wilting of mature plants are species of Fusarium, Verticillium, Cephalosporium, and Ceratostomella. It is now generally believed that in most cases wilting caused by fungi is due to toxins or to the plugging of the vessels by polysaccharides or other similar metabolic products of the fungus, rather than to plugging by the excessive mycelial growth in the vessels. Extracts of the mycelium or the culture filtrate of a number of these fungi cause effects that are the same as or similar to those caused by the fungi themselves in their respective hosts. A definite correlation between the pathogenecity of two strains of Fusarium lycopersici and the toxicity of their metabolic products was demonstrated by Haymaker (1928). There was similarity of symptoms and of the effect of temperature on wilting. The culture filtrate was more toxic when the fungus was grown at 28°C. than that obtained at any other temperature. The toxic substance was not identified. Other workers (Plattner and Clausson-Kaas, 1945; Woolley, 1946) have reported that the wilt-inducing compound produced by F. lycopersici is lycomarasmin, a peptide of aspartic acid. Gaumann and Jaag (1947) reported that clavacin exerted a wilting effect on detached tomato shoots similar to that of lycomarasmin. But, whereas lycomarasmin acted chiefly on the cells of the leaf blade, clavacin is toxic mainly to the phloem and paren- chyma of wood and cortex of the stem and petiole. The action of both compounds is believed to be similar, destroying the semipermeability of the plasma membranes, thereby decreasing the water-holding capacity of the cells and inducing wilting. Various polysaccharides have been shown to produce wilting in tomato cuttings (Hodgson et al., 1949). Since there was a direct relationship between molecular weight and wilt-inducing action of these compounds, it was concluded that their action was mainly by mechanically interfering with the transportation of water. Dimond (1947) also reported wilting of elm leaves due in part to a polysaccharide produced by Ceratostomella ulmi in culture. Its action is believed to be similar in naturally infected elm trees. More recently, Feldman et al. (1950) have presented evidence to show that the primary wilt-inducing agent produced by C. ulmi is not the polysaccharide, but a toxin. The production of toxin in liquid culture filtrate was greatly influenced by the pH of the medium, being greater in PARASITISM AND RESISTANCE 379 buffered media at pH 4.25 than at 5.25, although growth was more rapid in the less acid medium (Fig. 76). The toxin was shown to be irreversibly- inactivated at pH G or above. The introduction of calcium hydroxide into trees and the application of basic chemicals to the soil have been somewhat successful in retarding the disease. Presumably, these chem- icals act by raising the pH of the sap of the tree. Days Fig. 76. Growth of Ceratostomella ulmi and production of toxin, as measured by wilt of tomato seedlings induced by culture filtrate, in buffered media at different pH levels. Note that toxin production is favored by the more acid medium, while growth is greater in the less acid medium. (Courtesy of Feldman, Caroselli, and Howard, Phytopathology. 40: 348, 1950.) The varieties of oats susceptible to toxic culture filtrates of Helmin- thosporium victoriae were the same that were susceptible to the fungus in nature (Meehan and Murphy, 1947). Plants of Boone variety were killed, but Clinton plants were unaffected when grown in the same con- centrations of the filtrate. The toxic substance, which was not identified, was produced when the media contained either organic or inorganic nitrogen. This species differs from H. sacchari, which was reported by Lee (1929) to reduce nitrates to nitrites, which were toxic to sugar cane. 380 PHYSIOLOGY OF THE FUNGI The toxicity of the metabohc products of Fusarium vasinfectum was found to be dependent upon the medium on which the fungus was cultured (Rosen, 192G). Filtrates of cultures grown in a medium containing potassium nitrate and sucrose were highly toxic to cotton plants, while filtrates from cultures grown in a medium containing ammonium lactate, sodium asparaginate, and glycerin were not toxic. The filtrate of the nitrate-sucrose medium contained nitrites. Solutions of chemically pure sodium nitrite were also decidedly toxic to cotton plants. We may assume that the action of this fungus in converting nitrates to nitrites is the same within the host plant as it is in the culture vessel. Thus, there seems to be abundant evidence that the metabolic products, including enzymes and toxins, of a given fungus vary both in kind and in amount with the pH and composition of the culture medium. On the other hand, the evidence that the same situation exists in nature is extremely scarce. One may speculate, however, that the types of nutri- ents furnished by the host cells and the pH of the cell sap may also influ- ence the metabolic products of the fungus in the host plant. If this is true, a given fungus may find the nutrients and environment supplied by one host particularly favorable for the production of a disease-inducing toxin or enzyme. If the host is unable to inhibit the action of these substances, disease may result. The natures of both the pathogen and the host determine the severity of the disease. This hypothesis may help to explain, in part, the variation in intensity of parasitism of a fungus on its different hosts. While there is little evidence to support this idea at present, it is hoped that experimental work will be conducted to test its merits. The possibility that the presence of vitamins may affect pathogenicity has been suggested (Pehrson, 1948; Prasad, 1949). There is no evidence that deficiencies for vitamins are correlated with either parasitism or pathogenicity, and vitamin deficiency may be excluded as a factor leading to the parasitic habit. Likewise, there seems to be little or no correlation between the nitrogen requirements of fungi and the parasitic habit. Nonliving organic materials in nature are sources of vitamins and organic nitrogen just as are the living plants. For example, Ustilago striiformis, a highly parasitic fungus, is self-sufficient with respect to vitamins, and some isolates are capable of utilizing nitrate nitrogen, while Phycomyces hlakesleeanus, an obligate saprophyte, is deficient for thiamine and is unable to utilize nitrate nitrogen. Opposed to the destructive parasites discussed above are the balanced parasites, which, in general, have a strong power to penetrate mechan- ically but whose chemical actions on the host are relatively weak. Most of the filamentous balanced parasites produce intercellular mycelium, sending haustoria into the host cells. These serve as food-absorbing PARASITISM AND RESISTANCE 381 structures, but the exact mechanism of the transfer of food is not so well understood. The haustorium of the filamentous parasite is very similar in its behavior to the intracellular nonfilamentous parasite, being sur- rounded by the protoplasm of the host cell. Haustoria may be of several forms, simple and nearly spherical, coiled, and branched in various ways. Most cytologists agree that there is a cellulose wall, or sheath, around the older haustoria. It is presumably formed by the host cell and suggests a weak mechanism of defense against the invading parasite, yet it does not prevent the diffusion of soluble food into the haustorium. The haustorium commonly comes into contact with the nucleus of the host cell. In 23 of the 35 cases (host-parasite combinations) reported (Rice, 1927, 1935), habitual contact was observed between haustorium and nucleus. Two theories as to the meaning of this contact have been suggested. One is that the haustorium seeks out the region of the cell nucleus in order to facilitate the absorption of food from the cell. The second theory is that the action of the cell nucleus is defensive and that in some cases it may cause the death and degeneration of the haustorium. In the case of Synchytrium (Chrysophylyctis) endohioticum the swarm cells migrate into close proximity w^th the nucleus of the host cell (Orton and Kern, 1919). In the majority of cases the nucleus is engulfed at the time or soon after the swarm cells unite to form the vegetative body of the parasite. The host nucleus disappears as the sporangia develop. The exact significance of this close relationship between parasite and host nucleus is not clear, but it apparently represents a more or less unique method of parasitism among the fungi. It is generally believed that the balanced parasite causes harm to a susceptible host primarily through its demand upon food and water. There is little or no evidence that the protoplast is attacked chemically, although host cells may be killed by growth pressure. There are numer- ous reports of the disappearance of food in the region of haustoria. Butler (1918) reported that starch is absent in cells containing haustoria of Sclerospora graminicola, and at the time of sporulation the host cells collapse and die. The only abnormal effect observed by Mains (1917) on the cells of corn parasitized by Puccinia sorghi was the absence of starch in the bundle sheaths near the rust pustules. He interpreted this to mean that the parasite uses the food materials before they reach the bundle sheath where they are normally stored. Similar disappearance of starch in the host cells near infection by Synchytrium endohioticum has been reported by Orton and Kern (1919). On the other hand, starch may accumulate in the infected tissues dur- ing early stages of development of rusts but usually disappears in later stages of development. This may be due to some disruption of the host's physiology. The physiological reactions of the host are known to involve 382 PHYSIOLOGY OF THE FUNGI translocation of food, transpiration, respiration, and photosynthesis. Increased respiration has been reported for some hosts, while a decrease has been found for others. The rate of transpiration is usually increased. An early infection of orange rust on Ruhus may even cause the formation of stomata on the upper epidermis, where they are normally lacking (Dodge, 1923). The reactions of the chloroplasts of the host cells are believed to indicate the degree of adjustment between the host and parasite (Rice, 1935). Local chlorosis and streaking are common symptoms of a number of diseases caused by haustoria-forming parasites. Thatcher (1939, 1942) has shown that certain obligate parasites cause an increase in permeability of the cell membranes of susceptible hosts. There was a decided reduction in osmotic pressure of the tissues of Pisum surrounding the rust hyphae. If the fungus is unable to bring about an increase in permeability so that it can obtain its necessary nutrients, the host is resistant. Thatcher found evidence that the plasma membranes of some resistant varieties of wheat may actually become less permeable as a reaction to the rust hyphae, and starvation of the fungus may result. The change in permeability incited by the balanced parasites seems to be similar to the action of the destructive parasites, except for the matter of degree. Thatcher (1939) believes that parasitism in the rusts has become highly specialized, and the intensity of the effect on permea- bility of the cell membranes has been reduced. The substance involved is apparently a metabolic product of the fungus. If the conditions afforded by a certain variety of host are favorable for the production of a comparatively large amount of toxin (assuming that this substance is a toxin) , the host cells may be killed and the further development of the obligate parasite would be prevented. The sudden death of the host cells is the condition described by Stakman (1914) as hyper sensitiveness. Hypersensitive hosts are highly resistant or immune to the pathogenic action of the obligate parasites. Stakman reported that, in varieties of wheat resistant to Pucciiiia graminis tritici, when the hyphae of the fungus come in contact with the host cells, the latter often show plasmolysis, disintegration, and finally death. After the death of a few surrounding cells the tips of the hyphae die. However, it was discovered that in some cases the hyphae may die before the host cells are killed. Stakman concluded that the problem of resistance to rvists is one of toxins of the parasite or the host, or both, and can best be explained by what he terms the "toxin or enzyme theory." Brooks (1948) also concluded that the death of the parasite is due to the lethal action of the host rather than to starvation. Opponents of the toxin (or enzyme) theory of parasitism in the rusts PARASITISM AND RESISTANCE 383 point out that no toxin has ever been demonstrated experimentally. Leach (1919) believes that each physiologic race of Puccinia graminis has its own characteristic food requirements which are met by only a few varieties of the host. According to this hypothesis, if a race of rust enters a host which does not meet its specific nutritional requirements, it dies, and enzymes which are injurious to the host cells are released. This hypothesis is supported by Wellensiek (1927) who worked with Puccinia sorghi. While it is evident that the food supply varies with the varieties of the host, it seems equally possible that the difference in nutrients may have a more indirect effect in determining whether the fungus survives. Is it merely that the fungus starves if the host does not provide the appropriate food, or are the conditions in the host unfavorable for the production of certain metabolic products which are essential to the pathogenic actions of the fungus? The type of host-parasite relationship found in Phyllachora graminis seems to be unique (Orton, 1924) . This fungus apparently has the power of digesting and absorbing the tissues within the leaf, producing cavities in which the ascocarps later form. The hyphae bore their way through the cell walls of any of these tissues and, in doing so, absorb a portion of the wall. The parenchyma cells become disorganized, and their contents disintegrate. The vascular cells may be invaded and partially absorbed and become filled with hyphae. The most striking physiological charac- teristic of this fungus is its ability to absorb, replace, and engulf the tissues of the host leaf without any external evidence of necrosis of the host. This would seem to indicate the presence of highly active cellulo- iytic enzymes (and perhaps others) confined to the area near the fungus, without the presence of toxic substances, which would cause necrosis of the leaf tissue. Actually, comparatively little is known about the activities which lead to parasitism, particularly of the balanced parasites. It is hoped that more planned experiments will be conducted in an attempt to gain more knowledge regarding the mode of parasitism of plant pathogens. Only by understanding the action of the parasite can we understand the basic facts underlying resistance and susceptibility. Parasitism and symbiosis with insects. There are numerous reports of the parasitic and symbiotic relations of fungi with insects. For a more complete discussion than this text offers, see Leach (1940) and Steinhaus (1946). In many cases the relationship is solely to the advan- tage of the fungus (true parasitism), but a number of cases of mutualistic symbiosis do exist. The fungi may be disseminated by the insects which serve as their hosts. One can only speculate regarding the basic nutri- 384 PHYSIOLOGY OF THE FUNGI tional requirements of these fungi, since very little is known. We may assume that rather specific nutritional needs, either for growth or for reproduction, are satisfied by the relation with insects. Among the fungi parasitic on insects the genus Entomophthora is the most common. Various common species attack houseflies, grasshoppers, and other insects. A direct correlation between the amount of precipita- tion and the number of infections on houseflies was reported by Yeager (1939). Massospora cicadina infects the seventeen-year cicada and produces spores inside the abdomen. The posterior portion of the abdo- men sloughs away, exposing the spores w^hile the insect is still able to crawl about. This is apparently the chief method of dissemination of the spores. The mode and time of infection are unknown. Species of Cordyceps are common on pupae and larvae of certain insects. The fact that C. militaris produces abundant mycelial growth on a variety of synthetic media in the laboratory suggests the possibility that in nature this fungus may grow on other substrata, requiring the insect association only to fruit. Fawcett (1910) described the use of a fungus, which he named Aegerita webberi, in controlling whitefly in the orange groves of Florida. Ascher- sonia aleyrodis has also been used for the same purpose. A chytrid, Myrophagus ucrainicus, is reported (Karling, 1948) as a parasite on scale insects in Bermuda, Louisiana, and Ontario. In severe outbreaks as many as 45 per cent of the female insects may be killed. It has also been transmitted to mealy bugs. Another group of fungi parasitic on insects is the Laboulbeniales. These are minute fungi developing almost entirely on the surface, sending short haustoria into the insect to obtain food. The symbiotic relationship between Septobasidium and scale insects is interesting because of the high degree of specialization on the part of the fungus (Couch, 1938) . The dependence of the fungus for its distribu- tion upon the migrating young scale insects was previously mentioned in Chap. 15 under Spore Dissemination. The fungus forms a crust over scale insects, some of which are parasitized while others are not. The uninfected females give rise to young insects, which may remain under the fungus crust, crawl out through tunnels under the fungus, or crawl out over the sporulation surface of the fungus. The young insects are infected only by the bud cells from the basidiospores, never by the older fungus hyphae. The bud cells germinate on the surface of the insect and apparently enter principally through the natural openings. The fungus then produces coiled haustoria, which absorb food directly from the circulatory system of the insect, which in turn sucks its food from the host tree. Some infected insects may settle down on the bark, while others crawl under a nearby protective fungus colony. Only the former are responsible for distributing the fungus, while the latter are responsible PARASITISM AND RESISTANCE 385 for the survival of the ah-eady formed fungus colonies. Connections are then made by anastomoses of the hyphae from the insect and the hyphae of the fungus crust under which the insect has come to rest. Thus, the fungus colony does not originate from one individual but from the aggre- gation of several individuals by anastomosis, or grafting. The parasitized insects are dwarfed and do not reproduce but may live as long as the uninfected insects. The fungus covers the insect's body but is in contact with it only by the numerous coiled haustoria. The insect in turn receives protection from severe weather conditions, from parasitic wasps, other insects, and birds. Certain species, particularly S. hurtii, are easily cultivated on liquid or agar media. Couch believes that failure of the fungus to fruit in culture may be due to lack of proper nutrition, which is furnished by insects in nature, or to a complicated heterothallic condi- tion of the fungus. Fungi parasitic on other fungi. The parasitic habit of many of the chytrids upon other aquatic or semiaquatic fungi and algae is apparently quite common. A number of these genera are described and illustrated by Fitzpatrick (1930) and Karling (1942). Practically nothing is known regarding their nutritional requirements. There appear to be fewer filamentous fungi parasitic upon fungi. The mention of only a few of these will serve as examples. Species of Piptocephalis, Chaetocladium, and Syncephalis are parasitic on other Mucorales. A number of fungi are reported to be parasitic on members of the Agaricaceae and other higher fungi. Among these are species of Spinellus, Mycogone, Hypomyces, Nyctalis, and some Myxomycetes. A species of Penicillium is parasitic upon an Aspergillus (Thorn and Raper, 1945). Of particular interest are the hyperparasites, fungi parasitic upon other parasitic fungi. Cicin- noholus cesatii is parasitic on the Erysiphales, and Darlucafilum is parasitic on Uredinales. So far as is known, no study of the basic nutritional requirements of these fungi has been attempted. Fungi parasitic on man and animals. Many of the fungi which cause disease of man and animals show distinct differences in morphology when grown under different conditions. The spore forms produced on artificial media may be quite different from those developed in the host. This may be a response to certain nutritional factors, to temperature differ- ences, or to the presence of certain chemical substances which inhibit or limit the production of certain spore forms. In general, the pathogens of man and animals have no unique nutri- tional requirements. Some are able to utilize inorganic nitrogen, while others are not; some are deficient for certain vitamins. Nickerson (1947) points out that there is no direct correlation between nutritional require- ments and pathogenicity. In fact, there is little concrete evidence regarding the mode of action of these fungi in causing disease. 38G PHYSIOLOGY OF THE FUNGI In the case of the dermatophytes, Nickerson has suggested that growth and sporulation in vivo may be affected by a chemical supplied to the hair and scales of the skin by diffusion from the adjacent resistant tissues. There is some evidence that resistance of skin to fungus infection may be influenced by the nutrition of the individual. For more complete discussions of the fungus diseases of man and animals, the student is referred to Nickerson (1947), Wolf and Wolf (1947), Conant et al. (1944), Emmons (1940), and Dodge (1935). The pioneering work of Sabouraud (1910) should also be consulted. Cultural characteristics and pathogenicity. Studies of numerous isolates of a given species or genus have indicated a possible correlation between pathogenicity and some particular cultural characteristic. The recognition of such relations and definite knowledge regarding them would be of great value to plant pathologists. One such study was made by Houston (1945) on 52 isolates of Corticium solani from various hosts. These isolates fell into three culture types based upon the characteristics of the mycelium and sclerotia. There was a certain degree of correlation between culture type and pathogenicity and symptoms on certain hosts. He concluded that the culture type of C. solani is more important in predicting the pathogenicity of an isolate than the host from which it was isolated. During a study of the physiological characteristics and pathogenicity of 143 isolates of Actinomyces, mostly from scabbed potato tubers, it was found (Taylor and Decker, 1947) that certain isolates produced a dark ring of growth at the surface of separated milk. This characteristic was correlated perfectly with the ability to produce typical scab lesions on potato tubers. No attempt was made to explain the basic relation of these two apparently unrelated physiological characteristics. RESISTANCE Resistance is the ability of a host to prevent or oppose the entrance or subsequent growth and development of a parasite. It may be effective either before or after penetration of the host. A host which cannot successfully prevent such actions of the parasite is susceptible. Studies in the nature of resistance have been only partially enlightening, and in many cases the nature of disease resistance is still obscure. Some of the present theories are based on what might be termed "circumstantial evidence," such as a general correlation between resistance and some characteristic of the host. There is sufficient evidence, however, that it is dangerous to generalize about the nature of resistance. It seems likely that in many cases the cause of resistance may be specific, being common perhaps to but one or only a few host-parasite combinations. The types of resistance may be placed for convenience into three PARASITISM AND RESISTANCE 387 groups: (1) mechanical, the prevention of penetration or of unlimited spread by the structure of the host; (2) functional, the prevention of penetration by stomatal action of the host; (3) physiological, chemical action against the parasite or incompatible food relations. The relative importance of these factors is difficult to determine, but Butler (1918) states that physiological characters are much more important as a factor for resistance than the anatomical characters of the host. Mechanical resistance might be considered as the first line of defense by the host. According to Melander and Craigie (1927) resistance of species of Berheris to infection by sporidia of Puccinia graminis is due to the thickness of the cuticle. B. thunbergii, which is immune, has a heavy layer of cutin, while in general the susceptible species have a thin layer. These conclusions were reached after anatomical studies and after using a mechanical device to measure the resistance of the epidermis to puncture. The thickness of the cuticle increases with age, as does the resistance to mechanical puncture and to infection. The same is true with the apple scab fungus and powdery mildew of barley; young leaves are susceptible but become more resistant with age. Resistance in some cases is apparently due to layers of cork cells formed by the host in advance of the invading parasite. Varieties of flax resistant to wilt {Fusarium lini) and of cotton to black root rot {Thielaviopsis hasicola) seem to be successful in walling off the parasite by forming such a layer of cork which it cannot penetrate. Varieties of potatoes resistant to scab {Actinomyces scabies) form cork more quickly when wounded than do susceptible varieties and are believed to owe their resistance to this characteristic. Thomas (1934) found that the newly formed layer of cork cells was penetrated by invading hyphae of Armillaria mellea and that the cork layer did not successfully stop the advance of this fungus. Brown (1936) states that there is some doubt as to whether the cork layer really functions at all or whether it is formed after the fungus has been stopped by some chemical means. Lignified tissues offer more mechanical resistance than nonlignified cells. Certain varieties of wheat resistant to stem rust have a compara- tively greater amount of sclerenchyma and a correspondingly lesser amount of collenchyma and parenchyma in the stem, as compared with susceptible varieties. The maturity of host tissue may be a factor in resistance, even though the tissue does not become lignified or suberized. Some of the systemic smut fungi in cereals are able to grow and penetrate the cell walls in meristematic tissue but are apparently unable to pene- trate the cellulose walls of mature parenchyma cells. After infection in the embryo or seedling stage, the fungus must continue to grow in the growing tip of the shoot if it is to reach the flower parts. Conditions which favor slow growth and delay the maturity of the host favor the 388 PHYSIOLOGY OF THE FUNGI fungus, while conditions which favor rapid maturity of the host cells may cause the fungus to be left behind in the mature tissues which it cannot penetrate. Hart (1929) studied the nature of resistance of wheat varieties to stem rust and described a type of resistance that she terms funciio7ial resistance, which is dependent upon the stomatal movements of the host, and con- cluded that the parasite enters the wheat only through open stomata. There has been frequent discussion regarding the importance of the acidity of the cell sap of the host and its effect upon resistance. The effects of cell-sap acidity may be threefold: (1) an increase in the hydrogen ions; (2) the toxicity of the organic acids; (3) the influence upon the chemical changes and the possible formation of toxic products by the host cells. In some cases these effects have not been satisfactorily distin- guished. Numerous examples may be found in the literature in which resistance has been attributed to the acidity of the host or host part. Butler (1918) refers to investigations showing that the leaves of varieties of grape resistant to powdery mildew contain three to five times as much acid as the nonresistant varieties. He also showed that the red rot fungus of sugar cane, Colletotrichum falcatum, was present in infected canes from sowing time but usually did not develop severely until matu- rity of the canes. He attributed this to either the increase in sugar or the decrease in acid. The more acid lemons are less attacked by the fruit- rotting fungi. The amount of acid in the fruit, as indicated by chemical analysis, may be greater than the amount necessary to check the growth of the fungus in culture (Cook and Taubenhaus, 1911). A number of workers have considered cell-sap acidity as a possible cause for resistance of wheat to stem rust, but this factor now is believed to be of little impor- tance. No correlation was found between resistance and acidity of the expressed sap (Hurd, 1924). Similarly, there was no correlation between resistance and hydrogen-ion values or the titratable-acid values of the juice of wheat plants resistant to Ustilago tritici (Tapke, 1929). Some of the most complete experimental evidence showing the correla- tion between acidity and resistance is presented by Reddy (1933) for different inbred lines of corn in relation to Basisporium gallarum. Briefly, he found that when the pH of the cob was below 5.0, resistance to cob infection was high. Resistance was notably lower at high pH values. Table 64 gives a summary of some of Reddy's experiments. Reddy also believes that the influence of pH may explain why the seedlings, which are acid, are resistant to infection by B. gallarum, while the dry kernels, which are neutral or alkahne, are susceptible. On the basis of evidence previously discussed, it is likely that the pathogenic activities of certain enzymes produced by B. gallarum are inhibited in media having pH of 5.0 or less. PARASITISM AND RESISTANCE 389 On the other hand, greater acidity of the cell sap may favor the develop- ment of some diseases. The susceptibility of certain varieties of grape to Guignardia bidwellii has been correlated with a greater amount of tartaric acid (Butler, 1918). This author points out that leaves are susceptible only while they are young and rich in tartaric acid. Table 64. Hydrogen-ion Readings of Apparently Healthy Cobs of 75 Inbred Lines of Corn and Incidence of Basisporium Ear Infection Following Both Natural and Artificial Inoculation (Redd3^ loim Agr. Expt. Sta. Research Bull. 167, 1933.) CobpH No. of inbreds in No. of in- No. of ears Ears class interval breds infected observed infected, % 4.4-4.7 5 0 116 0 4.8 6 1 121 2.5 4.9-5.0 14 7 312 7.4 5.1-5.2 16 12 313 22.7 5.3-5.4 12 11 258 38.0 5.5-5.6 7 7 175 41.7 5.7-5.8 8 7 185 33.5 5.9-6.3 7 6 173 48.6 According to Smith et al. (1946), there is evidence that slight variation in pH may have a greater influence upon disease resistance of a plant than is generally believed. Such resistance is not due directly to the number of hydrogen ions. These authors state: The observed behavior of hydroquinone and catechol, representatives of the widely occurring ortho- and para-dihydroxyphenolic compounds, suggested that hydrogen ion differences also may influence toxicity by affecting the rate or extent of conversion to the more toxic quinones on invasion by pathogens or by other injur5^ The possibility that the presence of the pathogen may alter the pH of the host cells, making it more favorable to extensive invasion, should not be overlooked. Apparently this situation exists in the relation of Erwinia carnegieana to its host, the giant cactus of Arizona. Boyle (1949) reported that the freshly expressed sap from uninfected plants gave pH readings of 5.0 to 5.5, while the healthy-appearing tissue from infected plants had pH values of 7.0 to 7.4, and the pH of discolored tissue not yet broken down was 8.7 to 9.0. These differences could not be attributed to genetic variation and were believed to be a result of the pathogen. The possibility that similar relations exist between fungus pathogens and their hosts seems to merit greater consideration than it has received. 390 PHYSIOLOGY OF THE FUNGI That resistance is due to the presence of some toxic substance, perhaps an organic acid or some related compound, in the living host cell is one of the most popular theories. However, detailed proof of the effective- ness of such a compound, even though present in the plant, is often diffi- cult to obtain. Cook and Taubenhaus (1911) list some organic acids in order of their toxicity as follows: tannic, gallic, malic, tartaric, and citric. They state that vegetable juices contain an enzyme which acts upon gallic acid to produce tannin or a tannin-like compound which is toxic to fungi. The amount of the enzyme decreases with maturity and ripening of the fruits (apples, pears, persimmons, etc.), which accordingly become more susceptible to rot fungi. An outstanding example of chemical resistance is that described by Link and Walker (1933) for onion smudge caused by CoUetotrichum circinans. The cell sap of the colored varieties (resistant) is much more toxic to the fungus than the cell sap of the white-skinned varieties (sus- ceptible) . Furthermore, the sap of the colored varieties contains catechol and protocatechuic acid in amounts that would account for the resistance of these varieties to the fungus. The action of volatile and nonvolatile antibiotics in the fleshy scales of the onion is believed to be a definite factor in relative resistance of onion varieties to C. circinans, Aspergillus niger, and Botrytis allii (Hatfield et al., 1948). Reynolds (1931) explains resistance of flax varieties to Fusarium lini as being due to the higher amounts of glucosides, which upon hydrolysis yield hydrocyanic acid. Similarly, the resistance of species of Solamim to Cladosporiuni fulvum is believed to be due to the presence of higher amounts of solanine (Schmidt, 1933; cited by Brown, 1936). Rochlin (1933) believes that there is a direct connection betw^een resistance of crucifers to clubroot and the amount of glucosides, which on fermentation give rise to pungent mustard oils. The isolation of 2-methoxy-l,4-naphthoquinone from Impatiens balsamina was reported by Little et al. (1948). This substance had a high antibiotic activity against several fungi and was nontoxic to tomato and bean plants. This may be an example of a naturally occur- ring antibiotic as a factor in resistance. Fontaine et al. (1947) suggest that tomatin may be a factor in the resistance of certain tomato varieties to Fusarium lycopersici. An interesting theory of resistance to obligate parasites is presented by Dufrenoy (1936). He divides the hosts into three groups: (1) highly resistant, (2) moderately susceptible, (3) extremely susceptible. He believes that, when a fungus enters the highly resistant host, it kills the cells it penetrates and that the death of these cells alters the metabolism of the surrounding cells, so that their cell sap becomes rich in phenolic compounds, which prevents the further invasion by the pathogen. In the moderately susceptible host the host cells and their living contents PARASITISM AND RESISTANCE 391 are so modified that they revert to the embryonic condition and may even divide. When the obhgate parasite enters the extremely susceptible host, it causes so little disturbance that, at least in the first stages of infection, the metabolism of the host is afTected but little or not at all. Walker and Link (1935) caution against jumping at conclusions regard- ing the importance of phenolic compounds as factors in resistance. They point out that . . . the mere piesence of phenolic substances in a host plant does not warrant the conclusion that they play a role in the resistance of that host to a given parasite or parasites. Toxic phenolic substances might be present in concen- trations so low that their inhibitory effects are negligible, or they might also be present in concentrations that have a stimulative effect. When a phenolic sub- stance with a specific toxicity toward a given organism is present in the host in an appropriate concentration, it may be regarded as a part of the disease resisting mechanism of that host. The four fungi studied by Walker and Link {Colletotrichum circinans, Gibber ella saubinetii, Botrytis allii, and Aspergillus niger) reacted quite differently to the various phenolic compounds. Protocatechuic acid inhibited C. circinans at 1/800 and retarded growth at 1/12,800, while it did not affect A. niger at 1/200. Colored onions containing this acid are resistant to C. circinans but quite susceptible to A. niger. The immunity of monocotyledonous plants to Phymatotrichum omniv- orum is due to certain unidentified toxic materials present in monocots but apparently absent in most or all dicots (Ezekiel and Fudge, 1938). Growth of the pathogen was prevented by the expressed juices from a number of monocots but not by juices of susceptible dicots. Ether fractions of monocot roots, or other underground parts, were highly potent against the pathogen, while similar extracts from susceptible dicot plants were uniformly nonpotent. In some other highly parasitic fungi the action of the fungus causes the death of the surrounding cells, which then prevents the further spread of the parasite. Leach (1923) found that in a highly resistant variety of bean the hyphae of Colletotrichum lindemuthianum seldom attack more than one or two cells of the host. Both the host cells and the fungus hyphae then die, and the entire cell contents are stained a reddish brown. In less resistant varieties the parasite attacks more host cells, but sooner or later the mycelium disintegrates. Leach interprets this as "a nutri- tional phenomenon," which results in death of the fungus by starvation, and the products of autolysis then kill and stain the host cells. It has been pointed out previously that certain fungi are able to pene- trate some plants but are then unable to establish themselves (Stakman, 1914; Jones, 1919; Salmon, 1905). These plants may be either closely 392 PHYSIOLOGY OF THE FUNGI related or unrelated to those which serve as the natural host of the fungus. In such cases the failure to cause disease may be due to unfavoraVjle nutritional relations. The theory of a toxin-antitoxin, or toxin-counter- toxin, between parasite and host has been suggested by a number of investigators (Ward, 1905; Marryat, 1907; Stakman, 1914; Allen, 1923; Walker, 1924) as a possible explanation for resistance to the rusts. Cytological studies of Puccinia graminis tritici infections of both susceptible and resistant varieties of wheat were made by Allen (1923), who concluded that secretions from the fungus stimulate the metabolic activities of the susceptible host to produce more food, while in the resistant host the same secretions cause disintegration and death of the host cells near the infection. More distant cells may be stimulated. The haustoria usually die soon after the host cells are killed. Leach (1919) believes resistance to P. graminis tritici and P. graminis tritici- compacti can best be explained on the basis of specific food requirements of the parasite and specific food production by the host. It was sug- gested that the injury to the host cells might be due to an excess in amount of enzymes stimulated by a limited supply of food in resistant hosts. Similarly, Wellensiek (1927) believes that this theory best explains the resistance of corn to strains of P. sorghi. He suggests that the difference between susceptibility and resistance is of a quantitative nature and that the amount of the specific nutrient determines resistance or susceptibility. Walker (1924) points out that resistance may be due to the action of a number of factors and that a clear understanding of resistance must be based upon a thorough understanding of parasitism. Walker's excellent discussion of the nature of disease resistance gives many references to the literature on this subject. Host nutrition and its effect on the development and severity of disease is a relatively new phase of study, and much more investigation is neces- sary before general conclusions can be drawn. The fungi vary widely in their reactions to differences in host nutrition, the type of parasitism apparently being a determining factor. The action on the pathogen is believed to be principally indirectly through the effects of nutrition on the host, although it is possible that some of the vascular parasites may be directly affected by the nutrients which pass through the xylem. An increase in the salt concentration of the nutrient solution increased the development of clubroot, w^hile it decreased the severity of cabbage yellows (Walker, 1946). The development of Fusarium wilt of tomato was affected in a way similar to cabbage yellows. More recently, Gallegly (1949) reported that the development of Verticillium wilt of tomato was reduced with a reduction in salt concentration of the balanced solution used to grow the tomato plants. Stakman (1914) and Ward (1902) came to the conclusion that deficiencies in nitrogen and phosphorus salts avail- PARASITISM AND RESISTANCE 393 able to the host had no appreciable direct effect upon the resistance to rusts. A summary of the work on the effect of soil nutrients and environ- ment upon resistance to disease has been presented by Wingard (1941). The carbon metabolism of a plant likewise influences resistance to certain rusts. Waters (1926) found that urediospores of Uroniyces fabae developed on detached leaves floating on 5 per cent sucrose solution in the dark, while none formed when leaves were floated on water. These observations were confirmed by Yarwood (1934) for rust and powdery mildew of clover. It follows that active carbon assimilation increases susceptibility of the host to the obligate parasites. Although the environmental factors are of great importance in deter- mining the resistance or susceptibility to a disease, their effects are usually upon the host and only indirectly upon the parasite. Abundant refer- ences on this subject can be found in the literature. The effect of tem- perature upon the metabolism and resistance of certain hosts may be illustrated by Gihherella zeae on wheat and corn (Dickson, 1923). Seed- ling infection of wheat was found to occur at high temperatures and of corn at low temperatures; i.e., the temperatures unfavorable to host development. In the germination of w^heat at low soil temperatures the starch of the endosperm is hydrolyzed more rapidly than the proteins, which results in abundant sugar but little available nitrogen for seedling growth. Thus, the cell walls are thickened and more resistant. At higher temperatures both starch and proteins are rapidly hydrolyzed; there is a greater supply of available nitrogen, and growth is more rapid. The cell walls remain longer in the pectic condition and are more suscepti- ble to attack. In corn the situation is reversed. At high temperatures, which favor the corn, the cell w'alls develop more rapidly and are more resistant. Sharvelle (1936) concludes that the resistance of flax to flax rust cannot be attributed to any single factor but probably results from a number of factors operating together. Doubtless, the same statement could be applied to many other diseases to which the nature of resistance is not well understood. SUMMARY Some of the different types of parasitism may be summarized as fol- lows: (1) The parasite produces extracellular enzymes, particularly pectinase, w'hich dissolves the middle lamellae of the host cells, allowing the cells to separate (rotting). This may or may not be accompanied by toxic substances but results in the death of the cells. The soluble food materials are then free to be absorbed by the fungus. The insoluble foods stored in the host cells may be digested by other extracellular enzymes. This type is illustrated by the rots of fruits and vegetables. 394 PHYSIOLOGY OF THE FUNGI (2) The parasite may produce toxic materials or other substances whi( ii may be active at some distance from the fungus, but it usually does not cause the rotting of the tissue. This is illustrated by a number of wilt diseases and by some others. (3) The third type depends upon a con- genial nutritional relationship between the parasite and the host cells. In susceptible hosts of this type there is little or no apparent effect upon the host cells. The resistant hosts may show a high degree of sensitivity to the parasite, which may result in the death of the invaded cells and starvation of the parasite. This type of parasitism is characteristic of the balanced parasites. The balanced parasite enters the susceptible host cell and establishes a compatible food relationship, absorbing the soluble nutrients elaborated by the host, without disturbing the metabolic activity of the host in the early stages. In this respect, the relationship of parasite and susceptible host represents the most highly specialized type of parasitism. The destructive parasites, as a rule, are strong producers of toxins and exoenzymes, while the balanced parasites must be quite weak in this respect. In many host-parasite relations studied, there is a change in the permeability of the host cells surrounding the invading hyphae. This is believed to be a direct response to substances secreted by the parasite. Increased permeability would allow greater diffusion of water and nutri- ents from the host cells to the parasite. The metabolic products of the fungi involved in parasitism are for the most part undetermined, but they are known to include toxins, enzymes, and polysaccharides. Since the kind and amounts of such products are known to vary with the composi- tion of the medium in the laboratory, it is believed that like variation may occur in different hosts in nature. The basis of resistance to disease may be mechanical, functional, or physiological. Some of the known or proposed causes of physiological resistance are (1) cell-sap acidity; (2) toxic substances of the host; (3) inhibition of the activity of certain enzymes of the parasite by the host ; (4) hypersensitiveness ; (5) incompatible nutritional relationship; (6) decreased permeability of the cell membranes of the host, resulting in partial or complete starvation of the parasite; (7) a combination of various factors acting together. The obligate parasites, principally the rusts, offer some challenging unsolved problems for the future students of parasitism. Probably the principal one involves the culturing of such fungi under controlled condi- tions on media of known composition. All of the many attempts to solve this problem have met with failure, yet few investigators doubt that it can be solved. The phenomenon of heteroecism among the rusts is of great interest from the standpoint of food relationships. For instance, sve must either assume that the wheat and the barberry furnish the same PARASITISM AND RESISTANCE 395 nutrients for Puccinia graminis tritici, and the white pine and Ribes for Cronartium ribicola, or that the nutrient requirements of the haploid mycelium are different from those of the diploid mycelium. Much more investigation is needed to increase our knowledge of possi- ble correlations between pathogenicity and metabolic products. This should lead to a better understanding of parasitism. The possible role of antibiotics occurring naturally in host plants as a factor in disease resistance has received some attention recently, but much more knowl- edge of this type is desired. Many of the problems of today may come nearer to solution with a clearer understanding of the enzyme systems of the parasitic fungi and the basic principles of specific enzymatic action. REFERENCES Allen, R. F.: Cytological studies of infection of Baart, Kanred and Mindum wheats by Puccinia graminis tritici forms III and XIX, Jour. Agr. Research 26 : 571-604, 1923. Allen, R. F.: A cytological study of Puccinia triticina physiologic form 11 on Little Club Wheat, Jour. Agr. Research 33: 201-222, 1926. ♦Arthur, J. C, F. D. Kern, C. R. Orton, F. D. Fromme, H. S. Jackson, E. B. Mains, and G. R. Bisby: The Plant Rusts, John Wiley & Sons, Inc., New York, 1929. Bessey, E. a.: a Textbook of Mycology, The Blakiston Company, Philadelphia, 1935. Boyle, A. M.: Further studies of the bacterial necrosis of the giant cactus, Phyto- pathology 39 : 1029-1052, 1949. *Brooks, F. T.: Host resistance to fungi, chiefly in relation to obUgate parasites, Proc. Roy. Sac. (London), Ser. B, 135: 180-186, 1948. Brown, W.: Studies in the physiology of parasitism. I. The action of Botrytis cinerea, Ann. Botany 29: 313-348, 1915. Brown, W.: Studies in the physiology of parasitism. VHI. On the exosmosis of nutrient substances from the host tissues into the infection drop, Ann. Botany 36: 101-119, 1922. •*Brown, W.: The physiology of the host-parasite relation, Botan. Rev. 2: 236-281, 1936. Brown, W.: Physiology of the facultative type of parasite, Proc. Roy. Soc. (London), Ser. B, 135: 171-179, 1948. Butler, E. J.: Immunity and disease in plants, Agr. Jour. India, Special Indian Sci. Congr. Issue, 1918 : 10-28. Caldwell, R. M., and G. M. Stone: Relation of stomatal function of wheat to invasion and infection by leaf rust (Puccinia triticina), Jour. Agr. Research 52: 917-932, 1936. *Chona, B. L. : Studies in the physiology of parasitism. XIII. An analysis of factors underlying specialization of parasitism, with special reference to certain fungi parasitic on apple and potato, Ann. Botany 46: 1033-1050, 1932. CoNANT, N. F., D. S. Martin, D. T. Smith, R. D. Baker, and J. M. Callaway: Manual of Clinical Mycology, W. B. Saunders Company, Philadelphia, 1944. Cook, M. T., and J. J. Taubenhaus: The relation of parasitic fungi to the contents of the cells of the host plants, Delaware Agr. Expt. Sta. Bull. 91, 1911. 396 PHYSIOLOGY OF THE FUNGI Couch, J. N.: The Genus Septobasidium, The University of North Carolina Press Chapel Hill, 1938. De Bary, a.: Ueber einige Sclerotinien uiid Sclerotien Krankheiten, Bolan. Zig. 44:377-474, 188G. Dickson, J. G.: Influence of soil temperature and moisture on the development of the seedling-blight of wheat and corn caused by Gibberella sauhinettii, Jour. Agr. Research. 23 : 837-870 1923. DiMOND, A. E.: Symptoms of Dutch elm disease reproduced by toxins of Graphium ulmi in culture. Phytopathology 37 : 7, 1947. Dodge, B. O.: Effect of the orange-rusts of Rubus on the development and distribu- tion of stomata, Jour. Agr. Research 25 : 495-500, 1923. Dodge, C. W.: Medical Mycology, The C. V. Mosby Company, St. Louis, 1935. DuFRENOY, J.: Cellular immunity, Am. Jour. Botany 23: 70-79, 1936. Emmons, C. W.: Medical mycology, Botan. Rev. 6: 474-514, 1940. EzEKiEL, W. N., and J. F. Fudge: Studies on the cause of immunity of monocotyle- donous plants to Phymatotrichum root rot. Jour. Agr. Research 56 : 773-786, 1938. Fawcett, H. S. : An important entomogenous fungus, ]\Iycologia 2: 164-168, 1910. *Fbldman, a. W., N. E. Caroselli, and F. L. Howard: Physiology of toxin produc- tion by Ceratostomella ulmi, Phytopathology 40: 341-354, 1950. Fitzpatrick, H. M.: The Lower Fungi, Phycomycetes, McGraw-Hill Book Com- pany, Inc., New York, 1930. Fontaine, T. D., G. W. Irving, Jr., and S. P. Doolittle: Partial purification and properties of tomatin, an antibiotic agent from the tomato plant. Arch. Biochem. 12 : 395-404, 1947. Gallegly, M. E. : Host nutrition in relation to development of Verticillium wilt of tomato. Phytopathology 39: 7, 1949. Gaumann, E. : Pflanzliche Infektionslehre, Verlag Birkhauser, Basel, 1946. Trans. by W. B. Brierly as Principles of Plant Infection, Hafner Publishing Co., New York, 1950. Gaumann, E., and O. Jaag: Die physiologischen Grundlagen des parasitogenen Welkens. II, III. Ber. schweiz. botan. Ges. 52: 132-148, 227-241, 1947. Hart, H.: Relation of stomatal behavior to stem-rust resistance in wheat. Jour. Agr. Research 39: 929-948, 1929. *Hatfield, W. C, J. C. Walker, and J. H. Owen: Antibiotic substances in onion in relation to disease resistance. Jour. Agr. Research 77: 115-135, 1948. Hawkins, L. A., and L. B. Harvey: Physiological study of the parasitism oiPythium debaryanum on potato tuber. Jour. Agr. Research 18: 275-297, 1919. Haymaker, H. H. : Relation of toxic excretory products from two strains of Fusariu m lycopersici to tomato wilt. Jour. Agr. Research 36: 697-719, 1928. HiGGiNS, B. B.: Physiology and parasitism of Sclerotmm rolfsii, Phytopathology 17: 417-448, 1927. Hodgson, R., W. H. Peterson, and A. J. Riker: The toxicity of polysaccharides and other large molecules to tomato cuttings. Phytopathology 39: 47-62, 1949. Houston, B. R.: Culture types and pathogenicity of isolates of Corticium solani, Phijtopathology 35: 371-393, 1945. Hurd, a. M.: The course of acidity changes during the growth period of wheat with special reference to stem-rust resistance. Jour. Agr. Research 27: 725-735, 1924. Jones, F. R. : The leaf-spot disease of alfalfa and red clover caused by the fungi Pseudopeziza medicaginis and Pseudopeziza trifolii, respectively, U.S. Dept. Agr. Bull. 759, 1919. Karling, J. S.: Plasmodiophorales, published by the author, New York, 1942. PARASITISM AND RESISTANCE 397 Karling, J. S.: Chj'trifliosis of scale Insects, Am. Jour. Botany 35: 246-254, 1948. Klotz, L. J.: Inhibition of enzymatic action as a possible factor in the resistance of plants to disease, Science 66: 631-632, 1927. KuNKEL, L. O.: A contril)ution to the life history of Spongospora subterranea, Jour. Agr. Research 4: 26.5-278, 1915. Leach, J. G.: The parasitism of Puccinia grnminis tritici Erikss. and Ilenn. and Puccinia graminis tritici-compacti Stak. and Piem., Phytopathology 9: 59-88, 1919. *Leach, J. G.: The parasitism of Colletotrichum lindemuthianum, Minn. Agr. Expt. Sta. Bull. 14, 1923. Leach, J. G.: Insect Transmission of Plant Diseases, McGraw-Hill Book Company, Inc., New York, 1940. Lee, a.: The toxic substance produced by the eye-spot fungus of sugar cane, Helmin- thosporium sacchari, Plant Physiol. 4: 193-212, 1929. *LiNK, K. P., and J. C. Walker: The isolation of catechol from pigmented onion scales and its significance in relation to disease resistance in onions, Joiir. Biol. Chem. 100: 379-383, 1933. Little, J. E., T. J. Sproston, and M. W. Foote: Isolation and antifungal action of naturally occurring 2-methoxy-l,4-naphthoquinone, Jour. Biol. Chem. 174: 335-342, 1948. Mains, P>. B. : The relations of some rusts to the physiology of their hosts, Am. Jour. Botany ^: 179-220, 1917. Marryat, D. C.: Notes on the infection and histology of two wheats immune to the attacks of Puccinia glumarum, yellow rupt, Jour. Agr. Sci. 2: 129-138, 1907. Meehan, F., and H. E. Murphy: Differential phytotoxicity of metabohc by-prod- ucts of Helminthosporium victoriae, Science 106: 270-271, 1947. Melander, L. W., and J. H. Craigie: Nature of resistance of Berberis spp. to Puccinia graminis, Phytopathology 17: 95-114, 1927. NicKERsoN, W. J.: Biology of Pathogenic Fungi, Chronica Botanica Co., Waltham, 1947. Orton, C. R.: Studies in the morphology of the Ascomycetes. I. The stroma and the compound fructification of the Dothideaceae and other groups, Mycologia 16: 49-95, 1924. Orton, C. R., and F. D. Kern: The potato wart disease, Penna. Agr. Expt. Sta. Bull. 156, 1919. Pehrsox, S. O.: Studies of the growth physiology of Phacidium infestans Karst., Physiologia Plantarum 1: 38-56, 1948. Plattner, p. A., and N. Clausson-Kaas: Ueber ein Welke erzeugendes Stoffwech- selprodukt von Fusarium lycopersici Sacc, Helv. Chim. Acta 28: 188-195, 1945. Prasad, N.: Variability of the cucurbit root-rot fungus, Fusarium (H ijpo7nyces) solani f. cucurbitae, Phytopathology 39: 133-141, 1949. *Reddy, C. S.: Resistance of dent corn to Basisporium gallarum Moll., Iowa Agr. Expt. Sta. Research Bxdl. 167, 1933. Reynolds, E. S.: Studies on the physiology of plant disease, Ann. Missouri Botan. Garden 18: 57-95, 1931. Rice, M. A.: The haustoria of certain rusts and the relation between host and pathogenes. Bull. Torrey Botan. Club 54: 63-153, 1927. *Rice, M. a.: The cytology of the host-parasite relations, Botan. Rev. 1: 327-353, 1935. Rochlin, E. J.: On the question of non-susceptibility of Cruciferae to Plasmodio- phora hrassicae Wor., Bull. Plant Protection, (U.S.S.R.), Ser. II, Phytopathology, Leningrad, 1933. (Abst. in Rev. Applied Mycol. 13: 140-141, 1934.) 398 PHYSIOLOGY OF THE FUNGI Rosen, H. R.: Efforts to determine the means by which the cotton wilt fungus, Fusarivm vasinfedum, produces wilting, Jour. Agr. Research 33: 1143-1162, 1926. Sabouraud, R.: Maladies du cuir chevelu. Vol. Ill, Les Maladies cryptogamiques, Les Teignes, Masson et Cie, Paris, 1910. Salmon, E. S.: On the stages of development reached by certain biological forms of Erysiphe in cases of non-infection, Neiv Phytologist 4: 217-222, 1905. Schmidt, M.: Zur Entwicklungsphysiologie von Cladosporium Jidvxim und liber die Widerstandfjihigkeit von Solanum racemigerum gegen diesen Parasiten, Planta 20: 407-439, 1933. Sharvelle, E. J.: The nature of resistance of flax to Melampsora lint, Jour. Agr. Research 53: 81-127, 1936. *Smith, F. G., J. C. Walker, and W. J. Hooker: Effect of hydrogen-ion concentra- tion on the toxicity to Colletotrichum circinans (Berk.) Vogl. of some carboxylic acids, phenols, and crucifer extracts. Am. Jour. Botany 33: 351-356, 1946. Smith, L. D. S.: Clostridia in gas gangrene. Bad. Revs. 13: 233-254, 1949. Stakman, E. C. : a study in cereal rusts. Physiological races, Minn. Agr. Expt. Sta. Bull. 138, 1914. Steinhaus, E. A.: Insect Microbiology, Comstock Publishing Company, Inc., Ithaca. 1946. Tapke, V. F.: Influence of varietal resistance, sap acidity and certain environ- mental factors on the occurrence of loose smut of wheat. Jour. Agr. Research 39:313-319, 1929. Taylor, C. F., and P. Decker: A correlation between pathogenicity and cultural characteristics in the genus Actinomyces, Phytopathology 37 : 49-58, 1947. Thatcher, F. S. : Osmotic and permeability studies in the nutrition of fungus parasites. Am. Jour. Botany 26 : 449-458, 1939. *Thatcher, F. S.: Further studies of osmotic and permeability relations in parasi- tism. Can. Jour. Research 20: 283-311, 1942. Thom, C, and K. B. Raper: A Manual of the Aspergilli, The Williams & Wilkins Company, Baltimore, 1945. Thomas, H. E.: Studies on Armillaria mellea (Vahl.) Quel., infection, parasitism and host resistance. Jour. Agr. Research 48: 187-218, 1934. Vasudeva, R. S. : Studies in the physiology of parasitism. XL An analysis of the factors underlying specialism of parasitism, with special reference to the fungi Botrytis allii and Monilia frudigena, Ann. Botany 44: 469-493, 1930. Walker, J. C: On the nature of disease resistance in plants, Trans. Wisconsin Acad. Sci. 21 : 225-247, 1924. *Walker, J. C. : Soil management and plant nutrition in relation to disease develop- ment. Soil Sci. 61 : 47-54, 1946. Walker, J. C, and K. P. Link: Toxicity of phenolic compounds to certain onion bulb parasites, Botan. Gaz. 96 : 468-484, 1935. Ward, M. : On the relations between host and parasite in the bromes and their brown rust, Puccinia dispersa Erikss., Ann. Botany 16: 233-315, 1902. Ward, M.: Recent researches on the parasitism of fungi, Ann. Botany 19: 1-54, 1905. Waters, C. W.: The relations of bean rust grown on leaves in solution. Papers Mich. Acad. Sci. 5: 163-177, 1926. Wellensiek, S. J. : The nature of resistance in Zea mays L. to Puccinia sorghi Schw., Phytopathology 17: 815-825, 1927. * Wingard, S. a. : The nature of disease resistance in plants, Botan. Rev. 7 : 59-109, 1941. PARASITISM AND RESISTANCE 399 Wolf, F. A., and F. T. Wolf: The Fungi, Vol. II, John Wiley & Sons, Inc., New York, 1947. WooLLEY, D. W. : Strepogenin activity of seryl glycyl glutamic acid, Jour. Biol. Chem. 166: 783-784, 1946. Yarwood, C. E. : The comparative behavior of four clover-leaf parasites on excised leaves, Phytopatholgoy 24 : 797-806, 1934. Yeager, C. C. : Empusa infections of the house-fly in relation to moisture conditions of northern Idaho, Mycologia 31: 154-156, 1939. CHAPTER 18 PHYSIOLOGICAL VARIATIONS AND INHERITANCE OF PHYSIOLOGICAL CHARACTERS Variation in the results of experimental work with fungi is of frequent occurrence; it is perhaps even more frequent than uniformity. Different investigators conducting the same experiments with the same species of fungus have often failed to obtain the same results. Such variation may be attributed to (1) genetic differences in the strains or isolates used, (2) slight nutritional differences in the experiments, or (3) differences in the physical environment. Examples of the second and third groups of factors have been pointed out frequently in the earlier chapters. A brief discussion of the genetic differences involving physiological expression and the general mode of inheritance of these factors (in so far as they are known) will be given. PHYSIOLOGICAL VARIATION Variation in physiological behavior of different species of fungi has been noted in the preceding chapters. The present discussion emphasizes the physiological variation within a species, i.e., between different isolates, strains, or races, which show little or no morphological difference. Nutritional requirements. Variations in the nutritional requirements of different isolates of the same species are numerous. Differences in vitamin requirements or in carbon and nitrogen utilization may serve as examples. Differences in deficiencies for one or more vitamins have been reported for different isolates of Fusarium avenaceuni (Robbins and Ma, 1941), Sclerotinia minor (Barnett and Lilly, 19-47), Saccharomyces cerevisiae (Leonian and Lilly, 1942; Burkholder and Moyer, 1942), Sordaria fimicola (Hawker, 1939; Barnett and Lilly, 1947a), and numerous others. For example, certain isolates of Sordaria fimicola from nature are totally deficient for biotin alone, while others are deficient for both biotin and thiamine. A somewhat different type of variation is reported by Thren (1941) for Ustilago nuda. The haploid mycelium showed no deficiency for vitamins, while the diploid mycelium required an external supply of thiamine or pyrimidine. The plus and minus strains were also found to differ in their nutritional requirements. 400 VARIATION AND INHERITANCE 401 Different isolates of Ustilago striiformis have shown strikingly different responses to sources of carbon and nitrogen (Cheo, 1949). The isolates from bluegrass segregated into two groups based on mycelial type, ''frag- menting" and "mycelial." The "fragmenting" type grew well only on media containing sucrose and organic nitrogen, while the "mycelial" type could utilize a number of sugars and nitrate nitrogen. Single-spore (haploid) isolates from the same fruit body of Lenzites trdbea collected in nature varied nearly fourfold in their ability to synthesize thiamine (Lilly and Barnett, 1948). Induced deficiencies for a number of vitamins and amino acids have been demonstrated by Beadle (1946) in mutants of Neurospora and by Bonner (1946) in mutants of Penicilliiim. The mutations were induced by exposure of spores of these fungi to ultraviolet and X-ray radiation. Mutants that showed deficiencies for thiamine and differences in nitrogen requirements were also reported for Aspergillus terreus (Thorn and Raper, 1945). One mutant differed from most species of Aspergillus in its inability to utilize nitrate nitrogen. Fries (1948) describes spontaneous mutations of Ophiostoma which yield the same strains and in the same proportion as those induced by X rays. These results lead us to conclude that similar mutations are the principal cause of variation in the isolates obtained from nature. Response to environment. Isolates of the same species frequently vary in their physiological responses to some environmental factors, among which are temperature and light. For example, isolates of Phytophthora infestans were found to vary in their resistance to high temperature (Martin, 1949). Of the eight isolates studied, four from Louisiana withstood exposure to 36°C. for 6 days, while three isolates from Minnesota were killed after 4 days and one isolate from New York was killed in less than 6 hr. at the same temperature. The presence of the high-temperature strain is believed to be responsible for the prevalence of late blight in Louisiana during the past few years. Houston (1945) found that, for one group of isolates of Corticiiim solani, the optimum temperature for growth was 24 to 25°C. and the maximum was 33°C. For two other groups the optimum and maximum temperatures were 28 to 29°C. and 40°C., respectively. The three groups also varied in growth rates. Variation in response to light is illustrated by Choanephora cucur- hitarum. This was indicated first by Wolf (1917) for two isolates. The isolate used by Christenberry (1938) produced conidia in continuous total darkness, while two isolates used in our laboratory failed to produce conidia in continuous darkness (Barnett and Lilly, 1950). Metabolic products. Both qualitative and quantitative variations in the metabolic products of different isolates of the same species are com- 402 PHYSIOLOGY OF THE FUNGI mon. Industries involved in the commercial production of alcohols, certain organic acids, and antibiotics are in constant search for higher yielding "strains" of the species in present use, as well as of other species of fungi. Such a search led to the discovery of PenicilHum chrijsogenum Q176 and its variants, which are high producers of penicillin. Brewer's yeast is said to grow in media with an alcohol content as high as 14 to 17 per cent, while the baker's yeast is checked in about 4 per cent alcohol (Wolf and Wolf, 1947). Both yeasts belong to the species Saccharomyces cerevisiae. A different type of variation, apparently linked with sexuality, is reported in Mucor racemosus (Harris, 1948). Here, the production of an undetermined antibiotic seems to be confined to the minus strain. Varia- tion in bioluminescence is reported for Panus stypticus (Macrae, 1942). The fruit bodies and mycelium of the strain found in North America are luminescent, while those found in Europe are not (Fig. 77). Variants of the same species commonly differ in pigment production (Christensen and Graham, 1934; Leonian, 1929). Mutants, or saltants, are commonly lighter in color than the parent type. Sporulating ability. Many investigators have noted the spontaneous development of nonsporulating cultures or sectors from a sporulating mycelium. Variation in abundance of spores produced by different isolates from nature is also common. For example, some of the species which illustrate this variability are Fusarium spp., Phytophthora spp., Phoma terrestris, Gibber ella zeae, Glomerella cingulata, Lenzites trabea, Monilinia fructicola, and Ustilago striiformis. Variations in fruit bodies of Cyathus stercoreus produced in culture are described by Brodie (1948). Variation in production of sclerotia has been observed in isolates of Sclerotinia trifoliorum by Kreitlow (1949) and of S. sclerotiorum in our laboratory. Pathogenicity. Variability in the metabolic products such as enzymes and toxins and in the ability to establish compatible food relations with the host may be of great importance in determining pathogenicity. Das Gupta (1936) discusses the pathogenicity as well as other characteristics of "saltants." Such soil-inhabiting fungi as Fusarium spp. are notorious for their variability in pathogenicity within a species. Species of Helmin- thosporium (Christensen, 1922; Dickinson, 1932) and Cortidum solani (Houston, 1945) are likewise highly variable. In the highly parasitic fungi, such as the smuts, rusts, and powdery mildews, there is a high degree of physiologic specialization of races. The determination of physiologic races is based on infection types of several varieties or species of the host. Dickson (1947) reports the existance of 189 known physiologic races of Puccinia graminis tritici and 128 physiologic races of P. rubigo-vera tritici. Genetic studies indicate VARIATION AND INHERITANCE 403 that the physiologic races may vary in Init a single gene and that they may arise by hybridization or by mutation. There is abundant evidence that the haploid and diploid stages of some fungi may differ in pathogenicity. The haploid phase of a number of Fig. 77. Panus stypticus grown on malt agar. A, diploid mycelium, 4 weeks old, from a pairing between a haplont of the luminous American form and a haplont of the nonluminous European form, photographed by reflected light; B, the same culture as A photographed by its own light; C, a 2-weeks-old pairing between a nonluminous haplont, on the left, and a luminous haplont, on the right, photographed by reflected light; D, the same pairing as C photographed by its own light. (Courtesy of Macrae, Can. Jour. Research, Sec. C, 20: 424, 1942.) smuts is apparently unable to cause infection, while the diploid mycelium is pathogenic. Since the haploid and diploid mycelia of the heteroecious rusts parasitize different hosts, we must conclude that they also differ in pathogenicity. 404 PHYSIOLOGY OF THE FUNGI INHERITANCE OF PHYSIOLOGICAL CHARACTERS The genetics of the fungi has been, in general, a neglected study. Numerous papers have appeared on the sexuality of the fungi, particu- larly with regard to the various sexual or compatibility groups in the Basidiomycetes. The sexuality of the Mucorales has been studied to a lesser extent. Genetic studies of morphological characters have been decidedly fewer. Perhaps this is due to the failure to recognize definite morphological differences betw^een individuals of opposite sex but of the same species. An equally great handicap to such studies lies in the difficulty in obtaining the perfect stage of many of the fungi which other- wise might be suitable. Studies dealing with inheritance of physiological characters (if sexuality is excluded) are comparatively few and recent. The basis of inheritance. The physical basis of inheritance is the gene, located at a specific position on a certain chromosome. In mitosis the chromosomes and their genes divide, and half of each goes to each daughter nucleus. With the exception of parthenogenesis, all perfect stages of the fungi arise as a result of the union of two nuclei. These two nuclei may arise from the same haploid individual (homothallism) or from separate haploid thalli (heterothallism). The union of the two haploid nuclei, each with a single set of chromosomes, initiates the diploid nucleus, or the syncaryotic stage, in which the chromosomes are paired. The syncaryotic stage in fungi is usually short in duration, being followed closely by meiosis, which involves the separation of the two chromosomes (and genes) of each pair. Certain pairs of chromosomes may separate in the first division, while others separate in the second. Therefore, two successive nuclear divisions are necessary to complete the reduction of all pairs of chromosomes (and likewise all the pairs of genes). In the Ascomycetes and the Eubasidiomycetes karyogamy and meiosis occur in the same cell, the ascus and the basidium, respectively. In the smuts and rusts, meiosis typically takes place in a promycelium, while kary- ogamy occurs in the teliospore. When a single pair of genes is considered, half the haploid ascospores or basidiospores carry one gene and half carry the other gene. Inheritance in the Ascomycetes. Some of the outstanding genetic work has been done by Dodge (1927, 1928) and others with Neurospora, by Ames (1934) and Doweling (1931) with Pleurage anserina, by Edgerton et at. (1945), Chilton and Wheeler (1949), and their associates with Glomerella, and by Lindegren (1945, 1948) and his colleagues with yeasts. Most of these investigations have been concerned primarily with sexual or morphological characters. The life cycle of Neurospora is shown diagrammatically in Fig. 78. Beadle and his associates have contributed much to our knowledge VARIATION AND INHERITANCE 405 of the inheritance of physiological characters in the Ascomycetes. Beadle (1946) believed that, if the ability to synthesize a certain amino acid or growth factor were due to the action of a single gene, it should be possible to modify the gene in such a way that the fungus could no longer syn- thesize that compound. Previous work of other geneticists with corn, Drosophila, and other organisms had shown that exposure to X rays or ultraviolet radiation caused mutations by either destroying the gene or modifying it so that it could no longer function normally. Beadle found that exposure of conidia of Neurospora crassa and A^. sitophila to X rays or ultraviolet rays had the similar effect of causing mutations that were Germinating a SCO spore- .-^ Germinating ascospore Conidia Conidia Protoperittiecium A — ~- Protoperithecium a Hypnal fusion Fig. 78. Diagram of life cycle of Neurospora. (Courtesy of Beadle, Am. Scientist 34 : 36, 1946, and Science in Progress, 1947. Published by permission of the Society of the Sigma Xi.) expressed in the inability of the fungus to synthesize vitamins, amino acids, and other essential metabolites. The 'Svild type" of Neurospora is deficient for biotin but is self-suffi- cient for all other vitamins and for its necessary amino acids. The conidia were exposed to the ultraviolet rays of a Sterilamp for such a time that most of the spores were killed. The spores were then sown over the surface of agar plates in such concentration as to give individual "colonies," which were isolated and allowed to grow. When these were transferred to a minimal medium, containing sucrose, nitrate, mineral salts, and biotin, the failure of an isolate to grow showed an induced variation from the wild type in its capacity to synthesize essen- tial metabolites. The variant cultures were then selected and crossed with the wild 406 PHYSIOLOGY OF THE FUNGI strain of the opposite sex to determine if the changes were inherited. The ascospores from these crosses were planted on both the minimal medium and a complete medium. The appearance of the deficiency in half of the cultures was considered as evidence that the change was of genetic origin; i.e., a mutation. Transfers of the mutant to four different media (minimal, with amino acids, with vitamins, and complete) then deter- mined whether the deficiency was for an amino acid or a vitamin. All media contained biotin. For a diagrammatic scheme of the procedure see Fig. 79. X-ravs or ultraviolet 0 -^ © © © - Coniolict (asexuoil spores) Wild fv/pe O J- ^ \.y Crossed with wild t^pe of opposite sex Frui+inq body I -